Chimeric antibacterial polypeptides

ABSTRACT

Provided herein are antibacterial compositions and methods of making and using the compositions.

FIELD OF INVENTION

The present invention provides methods and compositions to reduce growthof microbial colonies, including infections, and includes therapeuticcompositions, methods for treatment of infections, and methods foridentifying additional such compositions.

BACKGROUND OF THE INVENTION

Bacteria are ubiquitous, ecologically diverse, and find unusual nichesfor survival. They are present throughout the environment, e.g., soil,dust, water, and on virtually all surfaces.

Pathogenic bacteria can cause infectious diseases in humans, otheranimals, and plants. Some bacteria can only infect or cause problems fora particular host, while others have a broader host specificity, and cancause trouble in a number of hosts. Diseases caused by bacteria arealmost as diverse as the bacteria themselves, e.g., food poisoning,tooth decay, anthrax, general infectious diseases, and even certainforms of cancer.

Certain bacteria are normally innocuous, but become pathogenic at theappropriate opportunity, or become problematic upon introduction to anabnormal site or situation. Persons lacking effective immune systems aremost vulnerable, and certain bacteria use weakened hosts to proliferateand disperse throughout the population.

Antibiotics have revolutionized clinical medicine over the last halfcentury. Since the original discovery of antibiotic phenomenon, themechanism of action and development of this class of remarkabletherapeutic entities has made enormous progress. See e.g., Therrien andLevesque (2000) FEMS Microbiol Rev. 24:251-62: Durgess (1999) Chest115(3 Suppl):19S-23S; Medeiros (1997) Clin. Infect. Dis. 24(Suppl1):S19-45; Jones (1996) Am. J. Med. 100(6A):3S-12S; Ford and Hait (1993)Cytotechnology 12(1-3):171-212; and Liu (1992) Compr Ther 18:35-42.Antibiotics had about $32B worldwide sales in 2002.

Yet the widespread appearance of antibiotic-resistant bacteria hasemphasized the vulnerability of current antimicrobial treatments tobacterial adaptation. See, e.g., Walsh (1992) Antibiotics: Actions,Origins, Resistance Amer. Soc. Microbiol.; Cunha (1992) AntibioticEssentials (Physicians Press); Amyes (2003) Magic Bullets, LostHorizons: The Rise and Fall of Antibiotics (Taylor & Francis); Axelsen(2001) Essentials of Antimicrobial Pharmacology: A Guide to Fundamentalsfor Practice (Humana Press); and Mainous and Pomeoy (eds. 2001)Management of Antimicrobials in Infectious Diseases: Impact ofAntibiotic Resistance (Humana Press). Multiple resistance plasmid NDM-1has been reported (Kumarasamy et al. (2010) Lancet Infectious Diseases10:597-602; and Walsh et al. (2011) Lancet Infectious Diseases, EarlyOnline Publication, 7 Apr. 2011, doi:10.1016/S1473-3099(11)70059-7).

Thus, improved methods for decreasing bacterial growth and survival, orlimiting bacterial pathogenicity, find great utility, especially forantibiotic resistant bacteria, which are most commonly Gram-negative.Antimicrobial effects are applicable to environmental, local, topical,and particularly in vivo colonization. The present invention addressesthese and other significant issues.

BRIEF SUMMARY OF THE INVENTION

Provided herein are antibacterial chimeric polypeptides comprising acomponent for traversing the outer membrane of a Gram negative bacteria(i.e., a membrane traversing domain, or MTD) and a component fordegrading the bacterial cell wall (i.e., a muralytic domain, or MD).

Provided are chimeric polypeptides comprising an MD derived from one ofthe MD sources listed in Table A and an MTD derived from one of the MTDsources listed in Table B. In some embodiments, the chimeric polypeptidereduces CFU of a culture of Gram negative bacteria compared to anuntreated control culture. In some embodiments, 1-100 nmol of thechimeric polypeptide lyses at least 50% of 10⁷ Gram negative bacteria ina CFU drop assay. In some embodiments, the bacteria are selected fromthe group consisting of Pseudomonas aeruginosa, Klebsiella pneumoniae,Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii,Salmonella typhimurium, Salmonella infantis, Shigella, Proteusmirabilis, and Burkholderia thailandensis.

In some embodiments, the MTD comprises a sequence selected from thegroup consisting of: amino acids 16-39 of SEQ ID NO:4; amino acids242-264 of SEQ ID NO:15; amino acids 242-271 of SEQ ID NO:17; aminoacids 220-406 of SEQ ID NO: 19; amino acids 220-400 of SEQ ID NO:21;amino acids 220-885 of SEQ ID NO:23, and variants thereof with a DASprofile between 1.2-2.6. In some embodiments, the variant thereof has1-6 hydrophobic amino acids substituted with amino acids with ahydropathy score of −2 or lower. In some embodiments, the DAS profile ofthe MTD is under 2.5. In some embodiments, the DAS profile of thechimeric polypeptide is under 2.5. In some embodiments, the MTDcomprises a sequence selected from the group consisting of: amino acids16-39 of SEQ ID NO:4; amino acids 242-264 of SEQ ID NO:15; amino acids242-271 of SEQ ID NO:17; amino acids 220-406 of SEQ ID NO:19; aminoacids 220-400 of SEQ ID NO:21; amino acids 220-885 of SEQ ID NO:23; andvariants thereof with at least 80% (e.g., at least 85, 87, 90, 92, 95,98, or 99%) identity to a sequence selected from the group consistingof: amino acids 16-39 of SEQ ID NO:4; amino acids 242-264 of SEQ IDNO:15; amino acids 242-271 of SEQ ID NO:17; amino acids 220-406 of SEQID NO: 19; amino acids 220-400 of SEQ ID NO:21; amino acids 220-885 ofSEQ ID NO:23.

In some embodiments, the chimeric polypeptide comprises a sequenceselected from the group consisting of: SEQ ID NOs:9, 11, 13, 15, 17, 19,21, and 23, and variants thereof having at least 90% (e.g., at least 92,93, 94, 95, 96, 97, 98, or 99%) identity to SEQ ID NO:9, 11, 13, 15, 17,19, 21, or 23. In some embodiments, the variant thereof has 1-10 of thecysteine or methionine amino acids substituted with non-cysteine,non-methionine amino acids.

In some embodiments, the chimeric polypeptide comprises an MD comprisinga sequence of amino acids 737-875 of SEQ ID NO:2, or a variant thereofcapable of lysing chloroform treated Gram negative bacteria. In someembodiments, the variant thereof has at least 90% (e.g., at least 92,93, 94, 95, 96, 97, 98, or 99%) identity to amino acids 737-875 of SEQID NO:2. In some embodiments, the MD comprises a sequence of amino acids683-889 or SEQ ID NO:2 or a variant thereof having at least 90% (e.g.,at least 92, 93, 94, 95, 96, 97, 98, or 99%) identity to amino acids683-889 of SEQ ID NO:2. In some embodiments, 1-100 nmol of the MD lysesat least 50% of 10⁷ chloroform-treated Pseudomonas aeruginosa bacteriain a CFU drop assay. In some embodiments, the variant thereof has 1-7 ofthe methionine amino acids substituted with non-methionine amino acids.

In some embodiments, the chimeric polypeptide comprises an MD comprisinga sequence at least 90% identical to amino acids 737-875 of SEQ ID NO:2and an MTD comprising a sequence at least 80% identical to amino acids16-39 of SEQ ID NO:4. In some embodiments, the chimeric polypeptidecomprises a sequence having at least 95% (e.g., 96, 97, 98, 99, or 100%)identity to SEQ ID NO:11 or 13.

In some embodiments, the MTD and MD are joined by a linker comprising3-5 positively charged amino acids (e.g., R, H, K, and combinationsthereof). In some embodiments, the positively charged amino acids areconsecutive or in close proximity (e.g., with 1-3 interveningnon-positive amino acids). In some embodiments, the MD is flanked on theN- and C-terminal ends with 3-5 positively charged amino acids. In someembodiments, the chimeric polypeptide is attached to a PAG or PEGmolecule.

Further provided are antibacterial compositions comprising a chimericpolypeptide as described above. In some embodiments, the antibacterialcomposition comprises the chimeric polypeptide an agent that reducesoxidation. In some embodiments, the antibacterial composition is apharmaceutical composition comprising the chimeric polypeptide and apharmaceutically acceptable excipient.

Further provided are methods of treating a bacterial infection in anindividual, e.g., inhibiting bacterial cell growth and/or reducing thenumber of target bacteria in the individual. In some embodiments, themethod comprises administering a pharmaceutical composition comprisingthe chimeric polypeptide to the individual in an amount effective toinhibit cell growth of the target bacteria in the individual, e.g.,compared to an untreated control. In some embodiments, the methodcomprises administering a pharmaceutical composition comprising thechimeric polypeptide to the individual in an amount effective to reducethe number of target bacteria in the individual compared to the numberof target bacteria present prior to treatment. In some embodiments, thetarget bacteria (the bacteria infecting the individual) are selectedfrom the group consisting of Pseudomonas aeruginosa, Klebsiellapneumoniae, Escherichia coli, Klebsiella pneumoniae, Acinetobacterbaumanii, Salmonella typhimurium, Salmonella infantis, Shigella, Proteusmirabilis, and Burkholderia thailandensis.

Further provided are methods of reducing the number of target bacteriaor inhibiting bacterial cell growth in an environment comprisingapplying a chimeric as described herein to the environment. In someembodiments, the target bacteria are selected from the group consistingof Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli,Klebsiella pneumoniae, Acinetobacter baumanii, Salmonella typhimurium,Salmonella infantis, Shigella, Proteus mirabilis, and Burkholderiathailandensis. In some embodiments, the environment is a surface (e.g.,for food preparation or medical use), a pharmaceutical composition, amedical device, a water or liquid source, or a food product.

The present invention is based in part upon the recognition thatphage-encoded cell wall degrading activities, e.g., murein-degrading (ormuralytic) enzymes, which are the core of the phage lysis functions, arealso found as structural components of the phage virion where they canbe required for infection of the bacteria. Gram-negative bacteria arecharacterized by a thin peptidoglycan cell wall surrounded by an outermembrane, which is lacking in Gram-positive bacteria. While a muralyticenzyme can digest the thin peptidoglycan layer of a Gram-negative cell,the outer membrane typically prevents access of the muralytic activityfrom the outside medium. Linking an enzymatically active muralyticsegment (fragment) to an agent (entity) that provides for transfer ofthe segment across the outer membrane allows the enzymatic activity tocontact the peptidoglycan layer, leading to degradation of thepeptidoglycan layer. The failure of the peptidoglycan layer causes thecell to rupture due to the enormous osmotic pressure across the innercell membrane.

The invention provides a recombinant chimeric polypeptide comprising:

-   -   a) a segment comprising at least 20 amino acid matches to amino        acids 16-39 of BPI TMD; and    -   b) a plurality of distinct segments of a least 20 amino acids        exhibiting at least 85% identity to amino acids 683-898 of GP36,        which segments do not overlap.

In some embodiments, the segment of at least 20 amino acid matches is atleast 22, 23, or 24 matches. In some embodiments, the plurality numbersat least three. In some embodiments, the distinct segments include oneof at least 40 amino acids exhibiting at least 92% identity. In someembodiments, the chimeric polypeptide exhibits at least 25, 30, 35, 40,50, 55, 60, 75, 80, 85, 90, 95% or higher % muralytic activity onpurified peptidoglycan from a Gram-negative bacterial host as comparedto GP36. In some embodiments, the chimeric polypeptide exhibits at least25, 30, 35, 40, 50, 55, 60, 75, 80, 85, 90, 95% or higher % muralyticactivity on an intact Gram-negative bacterial host as compared to thepolypeptide of SEQ ID NO:9. In some embodiments, the polypeptideexhibits at least 85, 90, 92, 95, 96, 97, 98, 99, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 9. In some embodiments, thechimeric polypeptide comprises at least amino acids 16-39 of BPI TMD. Insome embodiments, the chimeric polypeptide comprises a sequence with atleast 85, 90, 92, 95, 96, 97, 98, 99, or 100% identity to amino acids683-889 or 737-875 of GP36. In some embodiments, the chimericpolypeptide comprises or consists of SEQ ID NO: 9.

In some embodiments, the chimeric polypeptide is substantially free ofother phage proteins; or is substantially free of other proteinaceousmaterials. In some embodiments, the chimeric polypeptide is combinedwith another antimicrobial agent, including an antibiotic ordepolymerase agent. In some embodiments, the chimeric polypeptide isadmixed with a pharmaceutical excipient or is in a buffered or sterilecomposition.

In some; exhibits a bacterial cell wall degrading activity selected frommuralytic, glucosamidase, muraminidase, amidase, or endopeptidaseactivity. In some embodiments, the cell wall degrading (e.g., muralytic)activity is effective on multiple Gram-negative bacteria strains. Insome embodiments, the cell wall degrading (e.g., muralytic) activity iseffective on a Pseudomonas, Burkholderia, or Escherichia bacteriastrain. In some embodiments, the cell wall degrading (e.g., muralytic)activity is effective on one or more strains described as a Klebsiella,Acinetobacter, Salmonella, Proteus, Neisseria, Moraxella, Hemophilus,Enterobacter, Serratia, Legionella, or Helicobacter species.

The invention provides an expression vector comprising an isolated orrecombinant nucleic acid encoding the chimeric polypeptides describedherein. In some embodiments, the invention provides a recombinant cellcomprising the expression vector. In some embodiments, the cell is aeukaryote or prokaryote cell, e.g., to express the nucleic acid toproduce or secrete the protein.

Methods are provided, e.g., of enzymatically degrading the cell wall ofa bacteria by exposing the bacteria to the chimeric polypeptidedescribed herein. In some embodiments, the method results in at least a50, 60, 70, 80 or 90% decrease in a sensitive bacterial population on asurface (e.g., hospital, work, or furniture, etc.). In some embodiments,the method comprises introducing the chimeric polypeptide into an animal(e.g., human or other mammal), and results in at least a 20% decrease inthe population of sensitive bacteria in a selected location in or on theanimal. In some embodiments, the chimeric polypeptide is contacted with(applied to) an animal surface or compartment (e.g., digestive tract).In some embodiments, the chimeric polypeptide is applied to(administered to) an individual to treat a bacterial infection of theskin, mucosal tissue, blood, urinary tract, respiratory tract, orgastrointestinal tract. In some embodiments, the method comprises usingthe chimeric polypeptide in order to inoculate (induce an immuneresponse in) an individual with dead or lysed bacteria (see, e.g.,WO2003/026690).

The invention also provides a chimeric polypeptide comprising: amuralytic domain comprising at least two non-overlapping segments of atleast 15 contiguous amino acids from amino acids 683-898 of SEQ ID NO:2, and an outer membrane translocation domain comprising amino acids16-39 of SEQ ID NO: 4, wherein the muralytic domain has cell walldegrading activity against a target bacterium. In some embodiments, themuralytic domain comprises a plurality of non-overlapping segments of atleast 15 contiguous amino acids from a virion associated muralyticactivity of a lytic phage. In some embodiments, the muralytic domaincomprises a plurality of non-overlapping segments of at least 15contiguous amino acids encoded by a prophage structural gene. In someembodiments, the muralytic domain is from a myoviridae or podoviridaephage. In some embodiments, the muralytic domain is derived from themuralytic domain used by the phage to infect a host cell

In some embodiments, the target bacterium is a Gram-negative bacterium(has a bacterial outer membrane. In some embodiments, the targetbacterium is a Pseudomonas, Escherichia, Burkholderia, Klebsiella,Acinetobacter, Salmonella, Proteus, Neisseria, Moraxella, Hemophilus,Enterobacter, Serratia, Legionella, or Helicobacter species. In someembodiments, the target bacterium is resistant to one or more commontherapeutically used antimicrobial agent (e.g., antibiotic resistant).

In some embodiments, the invention provides methods for treating abacterial infection in a subject in need of treatment (e.g. anindividual having a bacterial infection, or at risk of developing aninfection) comprising: administering a chimeric polypeptide as describedherein to the subject, thereby treating the bacterial infection (e.g.,reducing the number of target bacteria in the individual). The subjectcan be a medical or veterinary patient including a human, a primate, ashow animal, or a companion animal. In some embodiments, the methodresults in a decrease CFU of a target Gram-negative bacterial populationof at least 20, 25, 30, 50, 60, 70, 80 or higher % in the individual, orat least 50, 60, 70, 80, 85, 90, 95, or higher % in culture (or in an invitro application, e.g., on a surface). The invention further providesmethods of disinfecting a surface or liquid by contacting the surface orliquid with the chimeric polypeptide (or a composition thereof).

In some embodiments, the invention provides methods for producing thechimeric polypeptide comprising expressing a nucleic acid encoding thechimeric polypeptide, e.g., in a eukaryotic cell. In some embodiments,the nucleic acid is in an expression vector such as a plasmid.

In some embodiments, the invention provides a recombinant muralyticprotein comprising amino acids 16-39 of BPI TMD linked to a muralyticdomain, wherein the protein has bacterial outer membrane traversingcapacity (activity) and peptidoglycan degrading activity. In someembodiments, the muralytic protein has a peptidoglycan degradingactivity of 50, 55, 60, 75, 80, 85, 90, 95% or higher % on purifiedpeptidoglycan from a Gram-negative bacterium as compared to GP36. Insome embodiments the amino acids 16-39 of BPI TMD and the muralyticdomain are linked using a non-peptide bond. In some embodiments, themuralytic protein is a chimeric protein. Further provided are arecombinant nucleic acid that encodes the muralytic protein, and a hostcell comprising the nucleic acid. In some embodiments, the inventionprovides methods for killing a bacterium possessing an outer membrane,comprising contacting the bacterium with the muralytic protein.

In some embodiments, the invention provides a recombinant muralyticprotein comprising amino acids 737-875 of GP36 (or amino acids 683-889of GP36) linked to a membrane transfer domain, wherein the protein hasbacterial outer membrane traversing capacity and peptidoglycan degradingactivity. In some embodiments, the muralytic protein has bacterial outermembrane traversing activity of 50, 55, 60, 75, 80, 85, 90, 95% orhigher % as compared to BPI-TMD. In some embodiments the amino acids16-39 of BPI TMD and the muralytic domain are linked using a non-peptidebond. In some embodiments, the muralytic protein is a chimeric protein.Further provided are a recombinant nucleic acid that encodes themuralytic protein, and a host cell comprising the nucleic acid. In someembodiments, the invention provides methods for killing a bacteriumpossessing an outer membrane comprising contacting the bacterium withthe muralytic protein.

In some embodiments, the invention provides an isolated recombinantmuralytic protein comprising a muralytic domain (providing peptidoglycandegrading activity) linked to a membrane transfer domain (providingbacterial outer membrane traversing capacity). Further provided is arecombinant nucleic acid encoding the muralytic protein and a host cellcomprising the nucleic acid. The invention further provides methods forkilling a bacterium possessing an outer membrane by contacting thebacterium with the muralytic protein.

In some embodiments, the invention provides an antibody specific for thenew combination of a muralytic domain and a membrane transfer domain,e.g., that recognizes an epitope unique to the combined polypeptide. Insome embodiments, the antibody is specific for the polypeptide of SEQ IDNO:9, and polypeptides having at least 85, 90, 92, 95, or higher %identity to SEQ ID NO:9, but does not significantly bind to apolypeptide of SEQ ID NO:4 or a polypeptide of SEQ ID NO:5. In someembodiments, the invention provides methods to detect or isolate achimeric polypeptide as described herein comprising contacting theantibody with a liquid or surface comprising the chimeric polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DAS hydrophobicity plots of (A) P266 and (B) P275.Relative hydrophobicity is shown along the length of the protein, withthe N terminus on the left and C terminus on the right. Note the Cterminal spike for P266 that is reduced in the modified P275 protein.

FIG. 2 shows the TMHMM (TransMembrane using Hidden Markov Models)predictions for (A) P266 and (B) P275. While the program does notpredict a transmembrane domain for P275, the chimeric protein stilleffectively crosses the bacterial outer membrane, and kills bacteria atthe same level as P266.

FIG. 3 shows hydrophobicity measurements and transmembrane predictionsfor the E. coli transmembrane protein CydA using (A) DAS plot, (B)TMHMM, and (C) Kyte Doolittle predictions. The DAS plot show that the6^(th) and 7^(th) predicted TMDs have relatively high hydrophobicity,indicating that these domains can be targeted for amino acidsubstitution.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The peptidoglycan (murein) sacculus is an essential structural componentof the cell wall of most bacteria. Made of glycan strands cross-linkedby short peptides, the sacculus forms a closed, bag-shaped structuresurrounding the bacteria cytoplasmic membrane. The sacculus mustwithstand up to 25 atmospheres of osmotic pressure. The sacculus isflexible, allowing reversible expansion under pressure, which allowsdiffusion of even large protein molecules. See, e.g., Silhavy et al.(2010) CSH Persp. Biol., 2:a000414; Vollmer et al. (2008) FEMS Microbio.Revs 32:149-167; Bos et al. (2007) Ann. Rev. Microbiol. 61:191-214; andCosterton et al. (1974) Bact. Revs. 38:87-110.

Many antibiotics act on the peptidoglycan layer of a target bacteriaspecies. This structure is thus a critical component in the survival ofa bacterial target. Attack of the peptidoglycan is a rational strategyfor killing target bacterial hosts. Although the peptidoglycan layer istypically about 1-3 layers thick, the outer membrane serves as apermeability barrier that prevents externally applied muralytic enzymesfrom reaching their substrate.

The present invention links the muralytic function to a membranepermeability function to achieve a new entity. The chimeric (and linked)constructs described herein combine a peptidoglycan degrading enzymeactivity with a membrane traversing function. In some embodiments, thetraversing function is achieved with a protein segment which allows theprotein with muralytic activity to be transferred across the bacterialouter membrane. In some embodiments, the traversing segment itselfmediates a membrane transfer event, thereby moving the muralyticactivity from outside of the bacterial outer membrane to the inside, andallowing contact between the enzyme and its peptidoglycan substrate. Insome embodiments, the traversing segment takes advantage of anendogenous translocation system in the outer membrane by presentingearmark motifs which signal the system to import the molecule into theperiplasmic space. In some embodiments, the traversing segment directsthe muralytic polypeptide to the outer leaflet of the outer membrane,and the muralytic polypeptide flips from the outer leaflet of the outermembrane to the inner leaflet, thereby delivering the muralytic segmentto the peptidoglycan substrate.

II. Murein-Degrading Enzymes; Lysozymes and Lysins

Muralytic domains (also called catalytic domains herein) include, e.g.,lysozyme proteins (Salazar and Asenjo (2007) Biotechnol. Lett.29:985-94). Breakdown of the peptidoglycan structure occurs naturally inat least four contexts. One is biosynthesis of the structure itself; asthe bacterial cell grows and divides, it must necessarily must breakdown the structure. See, e.g., Vollmer (2008) FEMS Microbiol Rev.32:287-306; Scheurwater, et al. (2008) Int. J. Biochem. Cell Biol.40:586-91; Keep, et al. (2006) Trends Microbiol. 14:271-276, and Babaand Schneewind (1998) EMBO J. 17:4639-4646. There are additionalsituations when the cell itself must rearrange or modify structure whichwas synthesized earlier. These activities can be derived from thebacteria themselves. Second, eukaryotic hosts degrade the structure uponclearing of an infection, e.g., using mutanolysin or lysozymes. See,e.g., Callewaert and Michiels (2010) J. Biosci. 35:127-60; Harder, etal. (2007) Endocr. Metab. Immune Disord Drug Targets 7:75-82; andLichtman, et al. (1992) J. Clin. Invest. 90:1313-1322. These activitieswill typically be derived from eukaryote hosts in or on which thebacteria live or can colonize. A third area is in phage replication,where the phage typically employs an endolysin to release the replicatedphages and lyse the bacterial host cell. See, e.g., Srividhya andKrishnaswamy (2007) J. Biosci. 32:979-90; and Loessner (2005) Curr.Opin. Microbiol. 8:480-487. These activities will typically be found inthe bacteriophage genome. This is a lysis of the peptidoglycan layer ofcells from within. A fourth context is where phage infection requiresthat the peptidoglycan barrier be traversed, as described inPadmanabhan, et al. WO2007/130655. This is degradation of thepeptidoglycan layer from the exterior of the cell. These activities willbe found as a component of the phage virion, and will typically beencoded in the phage genome.

Each of these mechanisms involve some means to disassemble thepeptidoglycan structure. Thus, muralytic activities are found in genomesof eukaryotic hosts for bacteria, in bacteria genomes themselves, and inphage (and related prophages) which target bacteria as hosts. Muralyticdomains can be found by homology to any of these sources, andinformatics can be used to identify candidates genes with theirrespective canonical motifs.

Peptidoglycan “degrading activities” can be converted into highlyeffective bacteriocidal activities for use against Gram-negativebacterial pathogens under therapeutic conditions, and can includemuraminidase, glucosaminidase, amidase, or endopeptidase activities.Exemplary muralytic domains can be identified, incorporated intochimeric constructs to be delivered to the peptidoglycan substrate,produced, purified, and confirmed to have bactericidal activity againstbacterial hosts with an outer membrane. Recombinant constructscomprising such activities have significant advantageous properties asantimicrobial compositions and formulations. Many of the peptidoglycandegrading activities of the invention are directed to Gram-negativebacteria, or bacteria which possess an outer membrane, but others willhave target specificity which may include either or both Gram-negativeand Gram-positive bacteria. The peptidoglycan structures of the twotypes of bacteria share certain linkages and structures, which may besusceptible to a selected muralytic activity. Thus, muralytic domainswhich can hyrdrolyze shared linkages may have broader target range thanthose which do not.

An example of the linked polypeptides of the invention uses a fragmentcomprising a lysozyme domain from Pseudomonas phage P134, which isclosely related to phage phiKMV. The ORF36 in phage P134 thatcorresponds to that in phiKMV lyses Gram-negative bacterial cells whoseouter membrane has been removed. Contacting the construct to a varietyof different Gram-negative bacteria after the outer membrane was removedresulted in the cells being broken down. These results demonstrate thatthe peptidoglycans from different Gram-negative bacteria species aresusceptible to the muralytic activity.

Sequence homology searches identify various other similar domains whichcan be used as alternative sources for peptidoglycan degradingactivities. The small size of the polypeptides exhibiting theseactivities affords efficient large scale production. Accessibility torelevant cell wall target components, e.g., peptidoglycans, at thebacterial target is provided, as are pharmacological distribution uponin vivo administration.

Relevant muralytic activities can be found within the lysozyme-likesuperfamily, lytic transglycosylase (LT), goose egg white lysozyme(GEWL); the Superfamily C100442 containing Lysozyme_like domain, whichcontains several members including the Soluble Lytic Transglycosylases(SLT), Goose Egg-White Lysozymes (GEWL), Hen Egg-White Lysozymes (HEWL),Chitinases, Bacteriophage lambda lysozymes, Endolysins, Autolysins,Chitosanases. All these members are involved in the hydrolysis ofbeta-1,4-linked polysaccharides. The Cysteine Histidine dependentAmidohydrolase/Peptidase (CHAP) domain is found in phage endolysins andbacterial autolysins. Most proteins containing a CHAP domain function aspeptidoglycan hydrolases and are commonly associated with amidases. SeeBateman and Rawlings (2003) Trends Biochem. Sci. 5:234-237; andPritchard, et al. (2004) Microbiology 150:2079-2087. See also theCarbohydrate-Active enZYmes Database found at cazy.org. The CAZYdatabase describes the families of structurally related catalytic andcarbohydrate-binding modules (or functional domains) of enzymes thatdegrade, modify, or create glycosidic bonds. Another source forendopeptidases is the database from the website found atmerops.sanger.ac.uk/cgi-bin/clan_index?type=P. Table A provides anexemplary list of enzymes having peptidoglycan degrading activities thatcan be used in the present invention. Additional similar or analogousactivities may be found which are similarly annotated, sharecharacteristic motifs with, or are homologous to members of the list.

TABLE A Muralytic Domain (MD) sources Description Details Phage lysozymeLysozyme helps to release mature phage particles from the cell wall bybreaking down the peptidoglycan. The enzyme hydrolyses the 1,4-betalinkages between N-acetyl-D-glucosamine and N- acetylmuramic acid inpeptidoglycan heteropolymers of prokaryotic cell walls. C-typelysozyme/alpha-lactalbumin C-type lysozymes are secreted bacteriolyticenzymes that cleave the family peptidoglycan of bacterial cell walls.Structure is a multi-domain, mixed alpha and beta fold, containing fourconserved disulfide bonds. Gene 25-like lysozyme This family includesthe phage protein Gene 25 from T4 which is a structural component of theouter wedge of the baseplate that has acidic lysozyme activity A1propeptide Most eukaryotic endopeptidases (Merops Family A1) aresynthesised with signal and propeptides. The animal pepsin-likeendopeptidase propeptides form a distinct family of propeptides, whichcontain a conserved motif approximately 30 residues long. Prophageendopeptidase tail This family is of prophage tail proteins that areprobably acting as endopeptidases. (e.g.: prophage tail protein gp18(NP_465809.1) from Listeria monocytogenes REPROLYSIN (M12B) FAMILY Themembers of this family are enzymes that cleave peptides. These ZINCMETALLOPROTEASE proteases require zinc for catalysis. Members of thisfamily are also known as adamalysins. Most members of this family aresnake venom endopeptidases, but there are also some mammalian proteinssuch as P78325 and fertilin. Virion associated muralytic enzymes fromphages (VAMEs) Mutanolysin Muramidase derived from Streptomycesglobisporus Peptidoglycan hydrolases eg: Mur-1 From EnterococcusN-acetylmuramidase, Mur-2 N- hirae ATCC9790 acetylglocosaminidase PhagePhiKMV gp36 Protein domain: Lysozyme like superfamily Phage LKD16 orf3Protein domain: Lysozyme like superfamily Phage LKD19 gp36 Proteindomain: Lysozyme like superfamily Phage phikF77 gp40 Protein domain:Lysozyme like superfamily Phage PT2 gp42 Protein domain: Lysozyme likesuperfamily Phage PT4 gp40 Protein domain: Lysozyme like superfamilyPhage 201 gp276 Protein domain: Lytic Transglycosylase (LT) or Goose EggWhite Lysozyme (GEWL) Phage F8 orf38 Protein domain: LyticTransglycosylase (LT) or Goose Egg White Lysozyme (GEWL) Phage 14-1 gp39Protein domain: Lytic Transglycosylase (LT) or Goose Egg White Lysozyme(GEWL) Phage LBL3 gp36 Protein domain: Lytic Transglycosylase (LT) orGoose Egg White Lysozyme (GEWL) Phage LMA2 gp38 Protein domain: LyticTransglycosylase (LT) or Goose Egg White Lysozyme (GEWL) Phage PB1 gp39Phage SN gp40 Protein domain: Lytic Transglycosylase (LT) or Goose EggWhite Lysozyme (GEWL) Phage phiKZ orf181 Protein domain: LyticTransglycosylase (LT) or Goose Egg White Lysozyme (GEWL)

III. Membrane Traversing Domains

To reach the interior and effectively infect a host cell, a phage mustcross the structural layers which surround the cell. In a Gram-positivebacterial cell, from the outside, these are the peptidoglycan layer andthe inner cell membrane. In a Gram-negative bacterial cell, there is anadditional lipid bilayer outer membrane surrounding the peptidoglycanlayer. This additional lipid bilayer forms another compartment betweenthe outer and inner membranes, which is the periplasmic space. See,e.g., Silhavy, et al. (2010) CSH Persp. Biol. 2:a000414; Bos, et al.(2007) Ann. Rev. Microbiol. 61:191-214; Nanninga (1998) Microbiol andMolec. Biol. Revs. 62:110-129; and Costerton, et al. (1974) Bacteriol.Revs. 38:87-110. The environment of the periplasmic space serves as anintermediate barrier. The peptidoglycan is typically much thinner in aGram-negative bacteria compared to a thicker peptidoglycan layer inGram-positive bacteria.

Because the bacterial outer membrane is a contiguous bilayer, it servesas a barrier to larger molecules accessing the periplasmic space. Theouter membrane is a selective semipermeable barrier that can protect thecell from harmful compounds in the environment, including antibiotics,and efficiently excludes larger proteins (e.g., a murlytic enzyme) fromthe periplasmic space.

A list of sources for transporting segments that can be used to effecttransport of an attached muralytic domain across the membrane includes:mammalian Bacterial Permeability Increasing protein (BPI); P134 andother holins; proteins P11 & P7 from PRD1 phage; TAMEs of Pseudomonasphages; Type VI secretion system in V. cholorae; holin-like protein(Tmp1) from (goat) skin surface; Apidaecin peptides 1a & 1b; phage P22tail spike protein; E. coli phage phiV10-putative tail fiber protein;hypothetical tail fiber Enterobacteria phage JK06; bacteriophage K1F (aT7-like phage); bacteriophage K1F (a T7-like phage withendo-N-acetylncuraminidase); T7 tail fiber protein-Enterobacteria phageT7; tail fiber protein Pseudomonas phage gh-1; and P2 gpH Enterobacteriaphage P2. See also, PDBTM, the first comprehensive and up-to-datetransmembrane protein selection of the Protein Data Bank (PDB). PDBTMdatabase is maintained in the Institute of Enzymology by the ProteinStructure Research Group at the website found at pdbtm.enzim.hu.Additional similar or analogous activities may be found which aresimilarly annotated, share characteristic motifs with, or are homologousto members of the list. Table B provides additional examples of membranetranslocation segments.

TABLE B Membrane Translocation Domain (MTDs) sources Description DetailsPhage holin 1 Phage proteins for bacterial lysis typically include amembrane- disrupting protein, or holin ABC_membrane ABC transporters areinvolved in the export or import of a wide variety of substrates rangingfrom small ions to macromolecules. They are found only in prokaryotesand their four constitutive domains are usually encoded by independentpolypeptides (two ABC proteins and two transmembrane domain (TMD)proteins) TonB The sequences in this set all contain a conservedC-terminal domain which is characteristic of TonB and is homologs. Aproline-rich repetitive region is found N-terminal to this domain; theselow- complexity regions are highly divergent and cannot readily bealigned. The region is suggested to span the periplasm. LolA TheLolA-lipoprotein complex crosses the periplasm and then interacts withouter membrane receptor LolB, which is essential for the anchoring oflipoproteins to the outer membrane. Mem_trans This entry represents amostly uncharacterised family of membrane transport proteins found ineukaryotes, bacteria and archaea. These proteins are typically 600-700amino acid residues long and exhibit 8-12 transmembrane segments. YojJYojJ is the N-terminus of a family of bacterial proteins some of whichare associated with DUF147 PF02457 towards the C-terminus. It is aputative membrane-spanning protein Mistic Mistic is an integral membraneprotein that folds autonomously into the membrane. It is conserved inthe Bacilli bacteria. The protein forms a helical bundle with a polarlipid-facing surface Trans membrane domain BPI is produced byneutrophils and is known to bind to OM of gram (TMD) of Bacterialnegative bacteria. A TMD was identified by bioinformatics analysis.permeability increasing protein (BPI) TMD from P134 holin Holins areknown to insert into the inner membrane(IM) of bacteria during phagelysis. The TMD is the region that is inserted into the IM. Purifyingholins is a challenge since the cells lyse 45 minutes after expressionand the holins get inserted into the membrane. Hence decided to use TMDof holins fused to GP36. Proteins P11 & P7 from The protein P11 & P7belongs to the DNA delivery apparatus of PRD1 phage PRD1. Afterattachment by the spike complex to the IncP encoded DNA transfercomplex, the receptor binding signals are transferred to the DNAdelivery apparatus and leads to conformational change in the PRD1vertex. This results in removal of spike complex and opening of thevertex which enables a tail to protrude containing proteins P11 &P7(Lytic transglycosylase). P11 has been located on the viral membraneand is strongly adhesive. It is believed that P11 interacts with OMfollowed by P7 which gains access to the peptidoglycan. Virionassociated muralytic Analysis & identification of potential OMtraversing molecules from enzymes (VAMEs) of VAMEs of bacteriophagesbacteriophages Antimicrobial peptides Anionic peptides, Linear cationicalpha helical peptides, Cationic peptides enriched for specific aminoacids, Anionic and cationic peptides that contain cysteines and formdisulfide bonds and Anionic and cationic peptides fragments of largerproteins. Bacteriocins S type Pyocins: Protease sensitive. S1, S2, S3 &AP41 cause cell death by DNA breakdown. S4 is predicted to have tRNaseactivity & S5, cytoplasmic membrane pore forming activity. Have 3domains, N terminal receptor binding domain, translocation domain & Cterminal killing domain. R & F type pyocins from Pseudomonas. Colicinsfrom E. coli; Channel-forming colicins (colicins A, B, E1, Ia, Ib, andN) are transmembrane domains that depolarize the cytoplasmic membrane.Have 3 domains, N ter translocation domain, binding domain & C terkilling domain. Initial binding is to porins and outer membraneproteins. Lipopolysacharide binding LBP binds to lipid A outer membraneon bacterial cells. LBP plays an protein (LBP) important role in theclearance of bacteria from the circulation that is mediated by CD14.Mannose binding protein Human MBP MBP selectively recognizes thecarbohydrate patterns (MBP) that decorate microorganisms such asbacteria, yeast, parasites, mycobacteria, and certain viruses. MBP doesnot recognize the sugars that decorate self-glycoproteins. Toll likereceptor (TLR) Are single, membrane-spanning, non-catalytic receptorsthat recognize structurally conserved molecules derived from MicrobesType VI secretion system in V. cholerae Holin like protein (Tmp1) fromgoat skin surface Apidaecin peptides 1a & 1b P22 Tail spike proteinRecognizes O antigen on surface of Salmonella; Has endo rhamnoseactivity (667 amino acids) E coli phage phiV10-putative LPS degradingactivity reported tail fiber protein Hypothetical tail fiberEnterobacteria phage JK06 Bacteriophage K1F, a T7-like phageBacteriophage K1F, a T7-like phage with endo-N- acetylneuraminidase T7tail fiber protein - Enterobacteria phage T7 Tail fiber proteinPseudomonas phage gh-1 P2 gpH Enterobacteria phage P2 HumanCathelecidins - hCAP18, LL37. Human defensins Mammalian lysozymesMammalian lactoferrin Mammalian lactoperoxidase Surfactant proteins Aand D (Collectins) produced by pulmonary cells Chemokine ligand 20(CCL20) produced by airway epithelial cells

In some embodiments, the translocating function can be achieved bychemical structure instead of a protein domain. As described above,alternative muralytic segments can have different efficiencies oftransfer across an outer membrane.

Rates of transfer across the outer membrane can be measured by a numberof methods. One method is to indirectly evaluate the results oftransfer, e.g., the effects of a muralytic segment reaching itsperiplasmic substrate. The criteria of measurement can be release ofmeasureable cell contents, substrate release, or cell lysis. Cellkilling can also be a surrogate measure of peptidoglycan digestion. SeeExamples section below describing binding of product to cell.

A more direct method is to track the number of molecules transferredinto the periplasmic space, e.g., using a detectable label. Theefficiency of transfer of a particular transfer segment will often beevaluated by measuring an amount of passenger segment transferred. Adetectable label can be used to differentiate between the periplasmicspace conditions (more oxidizing than outside the OM) and theextracellular environment See Rajarao et al. (2002) FEMS MicrobiologyLetters 215:267-272.

An efficient membrane transfer segment will effect at least a 3 foldincrease in the level of killing of target host by the muralytic domain,or at least a 3-fold increase in the level of transfer, as compared toabsence of the membrane transfer segment. In some embodiments, themembrane transfer segment will increase the level of killing or transferby at least 5, 7, 10, 15, 20, 30, 50, 80, 100, 150, 250 or more foldcompared to the absence of the membrane transfer segment. The assay istypically carried out under conditions which approximate theconcentrations which might be used according to the application. Theassay will typically measure transfer over a time period ranging fromminutes, e.g., 1, 2, 5, 10, 15, or 30 minutes, to an hour or two.

IV. Linkers Connecting Segments; Chemical Conjugation

The invention includes chimeric proteins which comprise two distinctdomains from heterologous sources. In some embodiments, the two domainsare part of a single polypeptide as a contiguous (chimeric) protein. Thetwo segments can be connected in either order, with the muralytic domainN-proximal to the membrane transfer domain (MTD) or vice versa. Thesegments can be linked directly or with a linker (peptide ornon-peptide). On occasion, the term transmembrane domain (TMD) will beused. The function may be more passive in the biophysical features ofthermodynamic interaction of the peptide with the hydrophobic membranebilayer, or may be interacting with an active process of serving as asegment which interacts with an active transport process, e.g., as therecognition component of an active transport mechanism which transfersthe entity from outside to inside the bacterial outer membrane of aGram-negative bacteria.

In some embodiments, the MTD can transfer the muralytic segment acrossthe inner membrane of a prokaryotic production host. In someembodiments, the MTD does not transfer the MD across the inner membrane,while retaining MTD activity for the outer membrane of a Gram negativebacteria. In some embodiments, the constructs described herein can beproduced instead in a eukaryotic cell system.

In some embodiments, the component segments are produced separately andlinked chemically. In some cases, synthetic polymerization methods areused to add peptides to existing sequences.

Chemical linkages or bioconjugation technologies may be used. See, e.g.,Niemeyer (ed. 2010) Bioconjugation Protocols; Strategies and Methods(Methods in Molecular Biology) Humana Press; Hermanson (2008)Bioconjugate Techniques (2d ed.) Academic Press; Lahann (ed. 2009) ClickChemistry for Biotechnology and Materials Science Wiley; Rabuka (2010)“Chemoenzymatic methods for site-specific protein modification” CurrOpin Chem Biol. 14:790-96. Epub 2010 Oct. 26; Tiefenbrunn and Dawson(2010) “Chemoselective ligation techniques: modern applications oftime-honored chemistry” Biopolymers 94:95-106; Nwe and Brechbiel (2009)“Growing applications of “click chemistry” for bioconjugation incontemporary biomedical research” Cancer Biother Radiopharm. 24:289-302;de Graaf, et al. (2009) “Nonnatural amino acids for site-specificprotein conjugation” Bioconjug Chem. 20:1281-95; the journalBioconjugate Chemistry (ACS); and Thordarson, et al. (2006)“Well-defined protein-polymer conjugates-synthesis and potentialapplications” Applied Microbiology and Biotechnology 73:243-254, DOI:10.1007/s00253-006-0574-4. For example, specific amino acids can beincorporated or added at either end, perhaps to constructs which haveremoved non-critical like residues, e.g., for cysteine residues.Accessible cysteine residues can be used to connect the segments bydisulfide linkages. Cysteine residues can also be linked withbifunctional maleimide linkers with thioether bonds. The linkers canalso have a hydrocarbon spacer of appropriate length, e.g., 6, 9, 12,15, 18, 21, 25, 29, 35, or more carbon chains.

Constructs were generated having accessible cysteines flanking the GP36CD lysozyme region, at either or both the N-terminus and C-terminus.These can be used to attach a variety of other chemical moieties. Longernon-peptide hydrophobic molecules can be attached to a Cys residue,including palmityl or similar groups, e.g., using an alkyl or acylhalide, e.g., palmityl bromide, or acyl chloride. The alkyl form willprovide a stable thioether linkage while the acyl form will provide aless stable thioester link. The alkyl or acyl hydrocarbon can integrateinto the outer membrane outer leaflet, which will flip to the innerleaflet at a detectable rate e.g., increasing with temperature.

V. Definitions

A “cell wall degrading activity” is an enzymatic activity that degrades,breaks down, disintegrates, or diminishes or reduces the integrity of abacterial cell wall (peptidoglycan layer). Unless indicated otherwise,e.g., by context, the term “muralytic” is used generically to mean “cellwall degrading.” Most wall degrading catalytic activities arehydrolytic. Thus, much of the terminology used refers to “muralytic”even if the catalytic mechanism does not involve hydrolysis. Degradationof defined or artificial substrates can be used to measure muralytic orstatic activity on a populational basis for the target. “Cell wallmuralytic activity” in a phage context is usually a characterizationassigned to a structure based upon testing under artificial conditions,but such characterization can be specific for bacterial species,families, genera, or subclasses (which may be defined by sensitivity).Therefore, a “bacterium susceptible to a cell wall degrading activity”describes a bacterium whose cell wall is degraded, broken down,disintegrated, or that has its cell wall integrity diminished or reducedby a particular cell wall degrading activity or activities. As explainedherein, other cell wall degrading activities originate from the hostbacterial cells, or on the phage structure (e.g., to serve inpenetration, but abortive to phage replication if destructive to thehost cell before intact phage are ready to be released). The structuresuseful in the penetration steps are particularly relevant to the presentinvention in that these activities operate on normal hosts from theexterior.

In some circumstances, a prophage sequence can be detected in abacterial genome. The prophage is often the remnants of an integratedphage genome which may have lost certain essential functions, and thusis embedded therein reflecting past biological function. See, e.g.,Kropinski, et al. (2007) Methods Mol. Biol. 394:133-75; Canchaya, et al.(2004) Mol Microbiol. 53:9-18; Canchaya, et al. (2003) Microbiol MolBiol Rev. 67:238-76; and Casjens (2003) Mol Microbiol. 49:277-300.Although a prophage can encode a substantial portion of the functions ofa lytic phage genome, the prophage normally does not pass through alytic cycle. Many of the structural components of a lytic phage haveequivalent or counterpart forms discoverable from a prophage sequence.Informatics analysis can typically determine the difference between asequence which once encoded the lytic activity used for infection ascompared to an endolysin activity used to lyse the target host cellafter phage assembly.

A “binding segment” refers to a targeting motif, which can recognizespecific structures on the bacterial outer surface. In Gram-positivebacteria, the outer surface of the bacteria is typically the mureinlayer (cell wall). Thus, a binding segment for Gram positive bacteriacan target a cell surface entity, e.g., protein, lipid, sugar, orcombination. Binding segments from lysozymes, endolysins, and such areknown and can be used. Other proteins which bind to bacteria include thePGRPs described below, the TLRs, flagellum and pill binding entities,and phage tail proteins involved in target recognition. In Gram-negativebacteria, the outer membrane presents various structures which can betargets for specific binding. The outer leaflet of the lipid bilayer orlipopolysaccharide can be exposed to the external environment.

A “membrane transfer domain (MTD).” also referred to as a TMD(transmembrane domain), translocating domain, transfer segment, and liketerms, refers to a molecular entity, e.g., a polypeptide domain orchemical entity, which can effect transfer of a linked muralytic segmentacross the outer membrane of a Gram negative bacteria. Such domain mayitself have the ability to translocate the associated segment across themembrane, or be recognized by an endogenous translocation system whichwill effect transport of the linked catalytic segment. The chimericpolypeptide can be transferred intact across the membrane, or bemodified during translocation. In some embodiments, the MTD does notsignificantly penetrate the inner membrane of an expression host. Insome embodiments, the MTD does not significantly penetrate the cellmembrane of a eukaryotic cell.

Although the outer membrane of Gram-negative bacteria protects cellsfrom many external agents, it is possible to weaken it specifically byvarious agents, collectively called permeabilizers, which help todisintegrate the LPS layer and increase the permeability of the OM tohydrophopic agents. Permeabilizers are compounds that weaken the OM andcan thus increase the activity of antimicrobials by facilitating entryof external substances capable of inhibiting or destroying cellularfunctions. This entry may be across the OM into the periplasmic spaceand perhaps ultimately into the cell cytoplasm. Permeabilizersthemselves may not be bactericidal, but may potentiate the activity ofother compounds, thus providing the possibility of synergistic action.The classical example of permeabilizers is the chelator EDTA, whichsequesters divalent cations that contribute to the stability of the OMby providing electrostatic interactions with proteins and LPS. Treatmentwith EDTA releases a large proportion of LPS from the OM, exposinghydrophobic phospholipids and creating a hydrophobic pathway for certainsubstances. This is noticeable as an increased susceptibility tohydrophobic agents. Permeabilsers may not be applicable in therapeuticcontexts since at high concentrations they are often toxic to cells. Inother contexts, they may be useful, e.g., in surface or devicesterilization applications. At lower concentrations they are able to actin permeabilising the outer membrane thus allowing access for moleculesto reach the peptidoglycan.

TABLE C Agents with outer membrane disrupting activity Designation AgentMode of action A Chelators 1 Ethylenediaminetetraacetic acid Removesstabilizing cations from the OM, notably Ca2+ and Mg2+. Releases LPS tothe external medium and creates a hydrophobic pathway. 2Na-hexametaphosphate Removes stabilizing cations from the OM, notablyCa2+ and Mg2. Increases sensitivity to hydrophobic antibiotics. 3Na2-pyrophosphate, Na-orthophosphate Destabilizes OM. Sensitizes cellsto nisin 4 Nitrilotriacetic acid Disintegrates the OM. Increasessensitivity to hydrophobic antibiotics. B Polycationic agents 1Polymyxins Displaces cations from the OM, causes membrane Tris (highconcentrations) damage. Binds to OM and increases sensitivity tohydrophobic antibiotics 2 Polymyxin B nonapeptide Permeabilizes the OMwithout significant release of LPS. Increases the cell surfacehydrophobicity. 3 Poly-L-ornithine, Poly-L-lysine Permeabilises the OMto hydrophobic antibiotics and releases LPS. 4 L-Ascorbate,Acetylsalicylate Destabilizes the OM 5 Lactoferrin, ransferring ReleasesLPS, increases sensitivity to rifampin 6 Cationic detergents, e.g.benzalkonium chloride Destabilizes hydrophobic interactions in OM 7Polyethyleneimine Intercalates in the OM and increases the membranesurface area without liberation of LPS-associated cell material.Sensitizes target cells to hydrophobic antibiotics and to detergents;causes the formation of vesicular structures on the surface of OM. CMembrane-perturbing proteins and peptides 1 Synthetic cationic peptidesDisorganization of LPS by interaction of the Cationic amphiphilicpeptides peptide with the anionic and hydrophobic lipid A D Terpenoidand phenolic compounds found in berries and herb plants 1 Thymol,carvacrol Destabilizes the OM and causes LPS release 2 Gallic acidDisplaces cations from the OM, causes membrane damage and LPS release. 3Phenolic berry extracts (cloudberry Displaces cations from the OM,causes membrane and raspberry) damage and LPS release. E Organic acidsand their salts 1 Citric acid cations from the OM, notably Ca2+ andMg2+, induces release of LPS. 2 Succinate, acetate, citrate Weaklyincreases membrane permeability F Other compounds 1 Chitosan (polymericβ-1,4-N- Binds to OM resulting in the loss of barrier acetylglucosaminefunction 2 Quinolones Low amounts (0.25 × MIC) Increases the sensitivityof Gram-negative bacteria to antimicrobial peptides by interacting withthe OM by removal of stabilizing divalent cations from LPS-bindingsites.

An “environment” of a bacterium can include an in vitro or an in vivoenvironment. In vitro environments can include a reaction vessel, e.g.,holding isolated or purified bacteria, a surface to be sterilized (e.g.,in a public health facility), equipment, surfaces in animal quarters, orpublic health facilities such as water, septic, or sewer facilities.Other in vitro conditions can provide mixed species populations, e.g.,including a number of symbiotically or interacting species in closeproximity. An in vivo environment can be a host organism infected by atarget bacterium. In vivo environments include organs, such as bladder,kidney, lung, skin, heart and blood vessels, stomach, fur, intestine,liver, brain or spinal chord, sensory organs, such as eyes, ears, nose,tongue, pancreas, spleen, thyroid, etc. In vivo environments includetissues, such as gums, nervous tissue, lymph tissue, glandular tissue,and biological fluids, e.g., blood, sputum, etc. Catheter, tubing,implant, and monitoring or treatment devices which are introduced intoor attached to the body may be sources of infection under normal usage.Environments also include the surface of food, e.g., fish, meat, orplant materials. Meats include, e.g., beef pork, chicken, turkey orother poultry. Plant materials include vegetable, fruits, or juices madefrom fruits and/or vegetables, or may include clothing or shelter. Insome embodiments, surfaces that have come in contact with abacterially-infected food product are treated with a protein of theinvention, including a VAME construct or chimera, e.g., GP36 CD segmentor P225. Sucrose and/or sorbitol may be useful to increases the osmoticpressure to make targets more susceptible to degradation ofpeptidoglycan layer.

“Introducing” a composition to an environment includes applying oradministering a compound or composition, and such that a targetedbacteria is exposed to the compound or composition. Introducing saidcompound or composition can be effected by live or dead bacteria whichmay produce or release such.

A “cell wall degrading protein” is a protein that has detectable, e.g.,substantial, degrading activity on an accessible cell wall or componentsthereof. “Muralytic” activity can be a result of the degrading activity.Exemplary degrading polypeptides include, e.g., GP36 CD segment or P225products, and functional structurally related entities, mutant andvariants thereof. Examples of cell wall degrading proteins are describedin the sequence listing, or derived, e.g., from phage phiKMV (seeNC_005045), or from the highly homologous ORF36 from phage phiKMV (seeGene ID 1482616; and NP_877475). Similar degrading domains can beidentified by motif analysis, their gene locations in the phage genome(or analogous prophage sequence), their structural location on the phage(or prophage counterpart) structure, e.g., tails or contact points ofnatural phage, similar motifs from mutated phage remnants (e.g.,pyocins), or encoded by prophage sequences. Cell wall degrading domainscan be derived, e.g., from the tail plates of myoviridae phage or endsof tails from siphoviridae phage, and other phage virion muralyticpolypeptides.

A “GP36 catalytic domain (CD) polypeptide” or grammatical variantthereof, refers to a polypeptide sequence exhibiting lytic(bacteriostatic) activity, typically encoded by the Pseudomonas phageP134 sequence highly homologous to ORF36 of phage phiKMV, or closelyrelated mutant or variant phage. SEQ ID NO: 1 provides the sequence of asegment of Pseudomonas phage P134 that is highly homologous to acorresponding ORF36 of the phage phiKMV. Exemplary variant ORF36polypeptides include polypeptide polymorphic variants, alleles, mutants,and interspecies homologs that: (1) have an amino acid sequence that hasgreater than about 60% amino acid sequence identity, 65%, 70%, 80%, 85%,90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity, preferably over one or more regions, e.g.,of at least about 8, 12, 17, 25, 33, 50, 65, 80, 100, 200, or more aminoacids, to an amino acid sequence encoded by an ORF36 nucleic acid fromPseudomonas phage P134 which is homologous to phiKMV, see, e.g.,Accession Number 1482616, (2) bind to antibodies, e.g., polyclonalantibodies, raised against a substantially purified immunogen comprisingan amino acid sequence of an active fragment of ORF36, andconservatively modified variants thereof; or (3) specifically hybridizeunder stringent hybridization conditions to an anti-sense strandcorresponding to a natural nucleic acid sequence encoding the ORF36polypeptide, and conservatively modified variants thereof; or (4) have anucleic acid sequence that has greater than about 65%, 70%, 75%, 80%,85%, 90%, or 95%, preferably greater than about 96%, 97%, 98%, 99%, orhigher nucleotide sequence identity, preferably over a region of atleast about 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, etc.,or more nucleotides, to the ORF36 encoding nucleic acid or a nucleicacid encoding fragment thereof. An example of a GP36 CD is the lysozymedomain running from amino acids 737-875 GP36. The nucleic acids andproteins of the invention include both natural or recombinant molecules.The full length ORF36 polypeptide and truncated fragments thereof can betested for degradative activity on cell wall components to determineboundaries critical for desired properties. In preferred embodiments,GP36 catalytic domain polypeptide has bacteriostatic activity againstvarious Pseudomonas, Escherichia, Kelbsiella, Acinctobacter, Salmonella,Proteus, Shigella, and Burkholderia bacteria. Some embodiments may alsoexhibit activity on Gram-positive bacteria, which lack the outermembrane. The concentration, time of action, temperature, and conditionsmay be optimized to have such activity on gram-positive targets.

Nucleic acids encoding cell wall degrading polypeptides can be amplifiedusing PCR primers based on the sequence of described cell wall degradingpolypeptides. For example, nucleic acids encoding GP36 CD polypeptidevariants and fragments thereof, as well as likely wall degradingactivity candidates, can be amplified using primers. See, e.g., Vybiralet al. (2003) FEMS Microbiol. Lett. 219:275-283. Thus, cell walldegrading polypeptides and fragments thereof include polypeptides thatare encoded by nucleic acids that are amplified by PCR based on thesequence of the identified cell wall degrading polypeptides. In apreferred embodiment, a bacteriostatic polypeptide or fragment thereofis encoded by a nucleic acid that is amplified by primers relevant tothe GP36 CD sequences described.

A “phage particle component” refers to, e.g., a head or tail componentof a phage, e.g., phage phiKMV. The invention provides that manydifferent phage types can be sources of the muralytic activity ascribedto the phage components. See, e.g., Piuri and Hatfull (2006) MolecularMicrobiology 62:1569-1585. Related sequences can be found in prophagesor incomplete phage genomes, typically found integrated into thebacterial host chromosome. Tail components typically mediate therecognition and attachment of the phage to the target host, and canpossess cell wall degrading activities which assist in penetration ofphage components into the host.

“GMP conditions” refers to good manufacturing practices, e.g., asdefined by the Food and Drug Administration of the United StatesGovernment. Analogous practices and regulations exist in Europe, Japan,and most developed countries.

The term “substantially” in the above definitions of “substantiallypure” generally means at least about 60%, at least about 70%, at leastabout 80%, or more preferably at least about 90%, and still morepreferably at least about 92%, 95%, 97%, or 99% pure, whether protein,nucleic acid, or other structural or other class of molecules.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalog refers to a compound that has the same basic chemical structureas a naturally occurring amino acid, e.g., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain a basic chemical structure as anaturally occurring amino acid. Amino acid mimetic refers to a chemicalcompound that has a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid.

“Protein”, “polypeptide”, or “peptide” refers to a polymer in which mostor all of the monomers are amino acids and are joined together throughamide bonds, alternatively referred to as a polypeptide. When the aminoacids are α-amino acids, either the L-optical isomer or the D-opticalisomer can be used. Additionally, unnatural amino acids, e.g.,β-alanine, phenylglycine, and homoarginine, are also included. Aminoacids that are not gene-encoded may also be used in the presentinvention. Furthermore, amino acids that have been modified to includeappropriate structure or reactive groups may also be used in theinvention. The amino acids used in the present invention may be the D-or L-isomer, or mixtures thereof. The L-isomers are generally preferred.In addition, other peptidomimetics are also useful in the presentinvention. For a general review, see, Spatola, A. F., in Weinstein, etal. (eds. 1983) CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES ANDPROTEINS, Marcel Dekker, New York, p. 267.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques. In particular, fusions of sequence may be generated,e.g., incorporating an upstream secretion cassette upstream of desiredsequence to generate secreted protein product.

A “fusion protein,” “chimeric protein,” “protein conjugate,” and liketerms refer to a protein comprising amino acid sequences that are inaddition to, in place of, less than, and/or different from the aminoacid sequences encoding the original or native full-length protein orsubsequences thereof. More than one additional domain can be added to acell wall muralytic protein as described herein, e.g., an epitope tag orpurification tag, or multiple epitope tags or purification tags.Additional domains may be attached, e.g., which may add additionalmuralytic activities (on the target or associated organisms of a mixedcolony or biofilm), targeting functions, or which affect physiologicalprocesses, e.g., vascular permeability or integrity of biofilm.Alternatively, domains may be associated to result in physical affinitybetween different polypeptides to generate multichain polymer complexes.

The term “nucleic acid” refers to a deoxyribonucleotide, ribonucleotide,or mixed polymer in single- or double-stranded form, and, unlessotherwise limited, encompasses known analogues of natural nucleotidesthat hybridize to nucleic acids in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated or by context, aparticular nucleic acid sequence includes the complementary sequencethereof.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of affecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes typically include at least promoters and/or transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors for effectingexpression can be included. In certain embodiments, an expressioncassette can also include nucleotide sequences that encode a signalsequence that directs secretion of an expressed protein from the hostcell. Transcription termination signals, enhancers, and other nucleicacid sequences that influence gene expression, can also be included inan expression cassette. In certain embodiments, a recombinant expressioncassette encoding an amino acid sequence comprising a muralytic activityon a cell wall is expressed in a bacterial host cell.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Modification of the heterologous sequence may occur, e.g., bytreating the DNA with a restriction enzyme to generate a DNA fragmentthat is capable of being operably linked to the promoter. Techniquessuch as site-directed mutagenesis are also useful for modifying aheterologous sequence.

The term “isolated” refers to material that is substantially oressentially free from components which interfere with the activity of anenzyme. For a saccharide, protein, or nucleic acid of the invention, theterm “isolated” refers to material that is substantially or essentiallyfree from components which normally accompany the material as found inits native state. Typically, an isolated saccharide, protein, or nucleicacid of the invention is at least about 80% pure, usually at least about90%, or at least about 95% pure as measured by band intensity on asilver stained gel or other method for determining purity. Purity orhomogeneity can be indicated by a number of means well known in the art.For example, a protein or nucleic acid in a sample can be resolved bypolyacrylamide gel electrophoresis, and then the protein or nucleic acidcan be visualized by staining. For certain purposes high resolution ofthe protein or nucleic acid may be desirable and, e.g., HPLC or massspectroscopy or a similar means for purification may be utilized.

The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence affectstranscription and/or translation of the nucleic acid corresponding tothe second sequence.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms or by visual inspection. In certainalignments of identity, no gaps are permitted, while in otheralgorithms, gaps are allowed with appropriate penalty measures.

The phrase “substantially identical,” in the context of two nucleicacids or proteins, refers to two or more sequences or subsequences thathave, over the appropriate segment, at least greater than about 60%nucleic acid or amino acid sequence identity, 65%, 70%, 75%, 80%, 85%,90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over one or more region of the sequencesthat corresponds to at least about 13, 15, 17, 23, 27, 31, 35, 40, 50,or more amino acid residues in length, more preferably over a region ofat least about 60, 70, 80, or 100 residues, and most preferably thesequences are substantially identical over at least about 150 residues,or over the entire length of the reference sequence. It will be notedthat three of the constructs specifically described have highhydrophobic stretches of 23, 23, and 30 amino acids, and data ispresented that at least 3 of 23 amino acid residues may be substitutedwith nonconservative residues while maintaining activity.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mot. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these and related algorithms (GAP,BESTFIT, FPASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (1995 andSupplements) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (ncbi.nlm.nih.gov) or similar sources.

A further indication that two nucleic acid sequences or proteins aresubstantially identical is that the protein encoded by the first nucleicacid is immunologically cross reactive with the protein encoded by thesecond nucleic acid, as described below. Thus, a protein is typicallysubstantially identical to a second protein, for example, where the twopeptides differ only by conservative substitutions. Another indicationthat two nucleic acid sequences are substantially identical is that thetwo molecules hybridize to each other under stringent conditions, asdescribed below.

The phrases “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind preferentially to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at each position where an arginine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Each polynucleotidesequence described herein which encodes a protein also describespossible silent variations, except where otherwise noted. One of skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and UGG which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule by standard techniques. Accordingly, each “silentvariation” of a nucleic acid which encodes a protein is typicallyimplicit in each described sequence.

Those of skill recognize that many amino acids can be substituted forone another in a protein without affecting the function of the protein,e.g., a conservative substitution can be the basis of a conservativelymodified variant of a protein such as the disclosed cell wall muralyticproteins. An incomplete list of conservative amino acid substitutionsfollows. The following eight groups each contain amino acids that arenormally conservative substitutions for one another: 1) Alanine (A),Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V), Alanine (A); 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T), Cysteine(C); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton (1984)Proteins).

Furthermore, one of skill will recognize that individual substitutions,deletions, or additions which alter, add, or delete a single amino acidor a small percentage of amino acids (typically less than 5%, moretypically less than 1%) in an encoded sequence are effectively“conservatively modified variations” where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

One of skill will appreciate that many conservative variations ofproteins, e.g., cell wall muralytic proteins, and nucleic acids whichencode proteins yield essentially identical products. For example, dueto the degeneracy of the genetic code, “silent substitutions” (e.g.,substitutions of a nucleic acid sequence which do not result in analteration in an encoded protein) are an implied feature of each nucleicacid sequence which encodes an amino acid. As described herein,sequences are preferably optimized for expression in a particular hostcell used to produce the cell wall muralytic proteins (e.g., yeast,human, and the like). Similarly, “conservative amino acidsubstitutions,” in one or a few amino acids in an amino acid sequenceare substituted with different amino acids with highly similarproperties, are also readily identified as being highly similar to aparticular amino acid sequence, or to a particular nucleic acid sequencewhich encodes an amino acid. Such conservatively substituted variationsof any particular sequence are a feature of the present invention. Seealso, Creighton (1984) Proteins, W.H. Freeman and Company. In addition,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence generally are also “conservatively modifiedvariations”.

The practice of this invention can involve the construction ofrecombinant nucleic acids and the expression of genes in host cells,preferably bacterial host cells. Optimized codon usage for a specifichost will often be applicable. Molecular cloning techniques to achievethese ends are known in the art. A wide variety of cloning and in vitroamplification methods suitable for the construction of recombinantnucleic acids such as expression vectors are well known to persons ofskill. Examples of these techniques and instructions sufficient todirect persons of skill through many cloning exercises are found inBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1999 Supplement) (Ausubel). Suitablehost cells for expression of the recombinant polypeptides are known tothose of skill in the art, and include, for example, prokaryotic cells,such as E. coli, and eukaryotic cells including insect (baculovirus),mammalian (CHO cells), fungal cells (e.g., yeast, Pichia, Aspergillusniger), and bacteriophage expression systems. Note that the N terminalMET is often removed in prokaryotic productions hosts. The presentlydescribed chimeric polypeptides include those with and without anN-terminal methionine on any or all of the peptide components.

Examples of protocols sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR), the ligase chain reaction (LCR), Qβ-replicase amplification andother RNA polymerase mediated techniques are found in Berger, Sambrook,and Ausubel, as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Ameim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3:81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sc. USA 86: 1173; Guatelli et al.(1990) Proc. Nat. Acad. Si. USA 87:1874; Lomell et al. (1989) J. Clin.Chem. 35:1826; Landegren et al. (1988) Science 241:1077-1080; Van Brunt(1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4: 560; andBarringer et al. (1990) Gene 89: 117. Improved methods of cloning invitro amplified nucleic acids are described in Wallace et al., U.S. Pat.No. 5,426,039.

VI. Commercial Applications

Various applications of the polypeptides described herein can beimmediately recognized. Many medical conditions result from bacterialinfections, described further in infectious disease and medicalmicrobiology textbooks. See, e.g., Kasper and Fauci (2010) Harrison'sInfectious Diseases McGraw-Hill Professional, ISBN-10: 0071702938,ISBN-13: 978-0071702935; Mandel (2008) Mandell, Douglas, and Bennett'sPrinciples and Practice of Infectious Diseases: Expert Consult PremiumEdition (7th Ed.) Churchill Livingstone, ISBN-10: 0443068399, ISBN-13:978-0443068393; Schlossberg (ed. 2008) Clinical Infectious DiseaseCambridge University Press, ISBN-10: 0521871123. ISBN-13:978-0521871129; Bauman (2011) Microbiology with Diseases by Body System(3d ed.) Benjamin Cummings, ISBN-10: 0321712714, ISBN-13:978-0321712714; and Murray, et al. (2008) Medical Microbiology (withStudent Consult Online Access, 6th ed.) Mosby, ISBN-10: 0323054706,ISBN-13: 978-0323054706. Therapeutic applications for these polypeptideconstructs will be appreciated.

The presently described outer membrane transversing, muralytic chimericproteins can be used for antibacterial treatment of articles which maybe contaminated in normal use. Locations, surfaces, equipment, orenvironments where target bacteria are public health hazards can betreated using the muralytic polypeptides described herein. Locations ofinterest include public health facilities where target bacteriacontaining materials exist. These materials may include waste products,e.g., liquid, solid, or air. Aqueous waste treatment plants mayincorporate the muralytic polypeptides to eliminate target bacteria fromeffluent, whether by treatment with the muralytic polypeptides or cellsthat express and release the muralytic polypeptides. Solid waste sitescan introduce the muralytic polypeptides to minimize possibility oftarget host outbreaks.

Food preparation areas and equipment can be regularly treated using themuralytic polypeptide compositions, thereby providing means toeffectively eliminate target bacteria. Medical and other publicenvironments subject to contamination can use similar means to minimizegrowth and spread of target microorganisms. The present methods can beused in contexts where elimination of target bacteria is desired,including air filtration systems, e.g., for an intensive care unit.

The chimeric muralytic proteins can be used as a protein stabilizer or apreservative, i.e., where the target bacteria are destabilizing agents.Such compositions can be used as part of the formulation for drugs, orpreservative for meat or other food products. In some embodiments, themuralytic polypeptides can be used in aquatic food products, e.g., as astabilizer or as a component of preservative formulations. Suchapplications are particularly useful for materials that must be keptantiseptic but cannot contain classical antibiotics.

Alternative applications include use in a veterinary or medical context.Means to determine the presence of particular bacteria, or to identifyspecific targets may utilize the effect of selective agents on thepopulation or culture. Inclusion of bacteriostatic activities tocleaning agents, including washing of animals and pets, may be desired.

The muralytic polypeptides described herein can be used to treatbacterial infections of, e.g., humans, animals, and plants. Themuralytic polypeptides can be administered to a subject prophylacticlyor where the subject has a bacterial infection. In addition, the presentmethods can be applied to display (e.g., zoo or performing), companion(e.g., dogs, cats, other pets), racing (e.g., horses), or farm (e.g.,dairy and beef cattle, sheep, goats, pigs, chicken, fish, shrimp,lobster, and the like) animals where the composition is applied toreduce the presence of bacteria. The muralytic polypeptides can be usedto treat infections caused by bacteria that replicate slowly, as thekilling mechanism does not depend upon host cell replication. Manycurrent antibacterial agents, e.g., antibiotics, are most useful againstreplicating bacteria. For example, the muralytic polypeptides can beused to target bacteria that replicate with doubling times of, e.g.,1-72 hours, 1-48 hours, 1-24 hours, 1-12 hours, 1-6 hours, 1-3 hours, or1-2 hours.

Medically relevant Gram-negative cocci species include Neisseriagonorrhoeae and spirochaetes (causing a sexually transmitted disease);Neisseria meningitides (causing meningitis); and Moraxella catarrhalis(causing respiratory symptoms). Relevant Gram-negative bacilli speciesinclude Homophiles influenzae, Klebsiella pneumonia, Legionellapneumophila, Burkholderia, and Pseudomonas aeruginosa (respiratoryproblems); Escherichia coli, Proteus mirabilis, Enterobacter cloacae,and Serratia marcescens (urinary problems), and Helicobacter pylori,Salmonella enteritidis, Salmonella typhi (gastrointestinal problems),and spirochaetes (sexually transmitted disease). Gram-negative bacteriaassociated with nosocomial infections include Acinetobacter baumannii,which cause bacteremia, secondary meningitis, and ventilator-associatedpneumonia, e.g., in intensive-care units of hospital establishments.

Other relevant that can be targeted using the present muralyticpolypeptides include Gram-negative species include Stenotrophomonas,Bdellovibrio, acetic acid bacteria, and alpha-proteobacteria such asWolbachia, the cyanobacteria, spirochaetes, green sulfur and greennon-sulfur bacteria.

Gram-variable organisms, which may have an outer membrane under certainconditions (display a Gram-variable pattern with Gram staining), canalso be targeted using the present muralytic polypeptides. Gram-variablebacteria include e.g., the genera Actinomyces, Arthobacter,Corynebactrum, Mycobacterium, and Propionibacterium, which have cellwalls particularly sensitive to breakage during cell division, anddisplay Gram-negative staining. In cultures of Bacillus, Butyrivibrio,and Clostridium, a decrease in peptidoglycan thickness during growthcoincides with an increase in the number of cells that stainGram-negative. In addition, the age of the bacterial culture caninfluence the results of the Gram stain.

VII. Administration

The route of administration and dosage of the muralytic polypeptidesdescribed herein vary with the infecting bacteria strain(s), the siteand extent of infection (e.g., local or systemic), and the subject beingtreated. The routes of administration include but are not limited to:oral, aerosol or other device for delivery to the lungs, nasal spray,intravenous (IV), intramuscular, intraperitoneal, intrathecal,intraocular, vaginal, rectal, topical, lumbar puncture, intrathecal, anddirect application to the brain and/or meninges. Excipients which can beused as a vehicle for the delivery of the therapeutic will be apparentto those skilled in the art. For example, the muralytic polypeptide canbe in lyophilized form and dissolved (resuspended) prior toadministration (e.g., by IV injection). The dosage is contemplated to bein the range of 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000,10000 or more muralytic polypeptide molecules per bacterium in the hostinfection. Depending upon the size of the protein, which may itself betandemly associated, or in multiple subunit form (dimer, trimer,tetramer, pentamer, etc.) or in combination with one or more otherentities, e.g., enzymes or fragments of different specificity, the dosemay be about 1 million to about 10 trillion/per kg/per day, andpreferably about 1 trillion/per kg/per day, and may be from about 10⁶killing units/kg/day to about 10¹³ killing units/kg/day.

Methods to evaluate killing capacity may be similar to methods used bythose of skill to evaluate intact replicating phage, e.g., plaqueforming units or pfu, though killing units may be better evaluated bydetermining the number of surviving bacteria after titration of thekilling units. Quantification of killing is distinct, sincenon-replicating phage will not form plaques on bacterial host lawns.Thus, serial dilution methods can be used to evaluate the quantity of“killing” units in place of standard pfu. Serial dilutions of bacterialcultures exposed to the killing compositions can be used to quantifykilling units. Total bacterial counts can be compared with viable colonyunits can establish the viable fraction of bacteria and what fractionwas susceptible to the killing constructs. Other means for evaluatingstasis activity may include release of intracellular contents, whethernatural or loaded, or enzymatic activity on defined or preparedsubstrates which correspond to natural cell wall structures.

The therapeutic(s) are typically administered until successfulelimination of the pathogenic bacteria is achieved. The inventioncontemplates single dosage forms, as well as multiple dosage forms ofthe compositions of the invention, as well as methods for accomplishingsustained release means for delivery of such single and multi-dosagesforms. Broad spectrum formulations can be used while specific diagnosisof the infecting strain is determined.

With respect to the aerosol administration to the lungs or other mucosalsurfaces, the therapeutic composition is incorporated into an aerosolformulation specifically designed for administration. Many such aerosolsare known in the art, and the present invention is not limited to anyparticular formulation. An example of such an aerosol is the Proventil™inhaler manufactured by Schering-Plough, the propellant of whichcontains trichloromonofluormethane, dichlorodifluoromethane, and oleicacid. Other embodiments include inhalers that are designed foradministration to nasal and sinus passages of a subject or patient. Theconcentrations of the propellant ingredients and emulsifiers areadjusted if necessary based on the specific composition being used inthe treatment. The number of enzyme killing units to be administered peraerosol treatment will typically be in the range of about 10⁶ to 10¹³killing units, e.g., about 10¹² killing units.

Typically, the killing will decrease the host replication capacity by atleast 3 fold, e.g., 10, 30, 100, 300, etc., to many orders of magnitude.Slowing the rate of host replication without killing can also havesignificant therapeutic or commercial value. Genetic inactivationefficiencies may be 4, 5, 6, 7, 8, or more log units.

VIII. Formulations

The invention further contemplates pharmaceutical compositionscomprising at least one cell wall degrading enzyme, e.g., muramidase, ofthe invention provided in a pharmaceutically acceptable excipient. Theformulations and pharmaceutical compositions of the invention thuscontemplate formulations comprising an isolated enzyme segment specificfor a bacterial host; a mixture of two, three, five, ten, or twenty ormore enzymes that affect the same or typical bacterial host; and amixture of two, three, five, ten, or twenty or more enzymes that affectdifferent bacterial hosts or different strains of the same bacterialhost, e.g., a cocktail mixture of enzymes that collectively inhibit thegrowth of multiple Gram-negative bacterial species. In this manner, thecompositions of the invention can be tailored to the needs of thepatient. The compounds or compositions can be sterile or near sterile.

A “therapeutically effective dose” is a dose that produces the effects,bacteriostatic (reducing bacterial growth) or bactericidal (killingbacteria), for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. See, e.g., Ansel, et al. (2010),Pharmaceutical Dosage Forms and Drug Delivery; Lieberman (1992)Pharmaceutical Dosage Forms (vols. 1-3), Dekker; Lloyd (1999) The Art,Science and Technology of Pharmaceutical Compounding; and Pickar (1999)Dosage Calculations. As is known in the art, adjustments for proteindegradation, systemic versus localized delivery, as well as the age,body weight, general health, sex, diet, time of administration, druginteraction, and the severity of the condition may be necessary, andwill be ascertainable by those skilled in the art.

Various pharmaceutically acceptable excipients are well known in theart. As used herein, “pharmaceutically acceptable excipient” includes amaterial which, when combined with an active ingredient of acomposition, allows the ingredient to retain biological activity andwithout causing disruptive reactions with the subject's immune system.Such excipients include stabilizers, preservatives, salt or sugarcomplexes or crystals, and the like.

Exemplary pharmaceutically carriers include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples include, butare not limited to, standard pharmaceutical excipients such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. In other embodiments, thecompositions will be incorporated into solid matrix, including slowrelease particles, glass beads, bandages, inserts on the eye, andtopical forms.

Further included are formulations for liposomal delivery, andformulations comprising microencapsulated enzymes, including sugarcrystals. Compositions comprising such excipients are formulated by wellknown conventional methods (see, e.g., Remington's PharmaceuticalSciences Chapter 43, 14th Ed., Mack Publishing Col). The proteins may besubjected to PEGylation to achieve advantages often deriving therefrom.See, e.g. Jevsevar, et al. (2010) Biotechnol. J. 5:113-128; Brocchini,et al. (2008) Adv. Drug Delivery Revs. 60:3-12; Jain and Jain (2008)Crit. Rev. Ther. Drug Carrier Syst. 25:403-47, PMID: 190626331; andShaunak, et al. (2006) Nature Chemical Biology 2:312-313. Alternativesexist for achieving similar stabilizing results. See, e.g.,Schellenberger, et al. (2009) Nature Biotechnology 27:1186-1192.

In general, pharmaceutical compositions can be prepared in variousforms, such as granules, tablets, pills, capsules (e.g., adapted fororal delivery), suppositories, microbeads, microspheres, liposomes,suspensions, salves, lotions and the like. Pharmaceutical grade organicor inorganic carriers and/or diluents suitable for oral and topical usecan be used to make up compositions comprising thetherapeutically-active compounds. Diluents known to the art includeaqueous media, vegetable and animal oils and fats. Formulations mayincorporate stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue.

The pharmaceutical composition can comprise other components in additionto the muralytic polypeptide, e.g., more than one active ingredient,e.g., two or more, three or more, five or more, or ten or more differentenzymes, where the different enzymes may be specific for the same,different, or accompanying bacteria. For example, the pharmaceuticalcomposition can contain multiple (e.g., at least two or more) definedwall degrading enzymes, wherein at least two of the enzymes in thecomposition have different bacterial host specificity or differentpeptidoglycan linkage specificity, such as a combination of atransglycolase and an endopeptidase. In this manner, the therapeuticcomposition can be adapted for treating a mixed infection of differentbacteria, or may be a composition selected to be effective againstvarious types of infections found commonly in a particular institutionalenvironment. A select combination may result, e.g., by selectingdifferent groups of cell wall degrading entities derived from variousbacteriophage of differing specificity so as to target multiple strainspresent, or potentially present in the infection. As noted above, thewall degrading enzyme can be administered in conjunction with otheragents, such as a conventional antimicrobial agent or a reagent whichprovides for efficacy against biofilm or capsule forming cultures.Various materials are described, e.g., in Davies and Marques (2009) J.Bacteriology 191:393-403; Kimura and Itoh (2002) Appl. and Env.Microbiology 69:2491-2497; Kim and Geider (2000) Phytopahtology90:1263-1268; Hughes, et al. (1998) J. Appl. Microbiology 85:583-590;and Bartell and Orr (1969) J. Virology 4:580-584. In some embodiments,an additive (e.g., fatty acid) or biofilm depolymerase may be added asan additional domain to the chimeric construct, as an additionalcomponent in a formulation, or administered in combination,simultaneously or sequentially, with the cell wall degrading activity.Active constructs based upon the described phage derived polysaccharidedepolymerases can be combined with the provided muralytic activities.

IX. Modification of Protein Sequence for Improved Production

MTDs are typically hydrophobic in nature, and a chimeric proteinincluding such an MTD may be insoluble when expressed in a productionhost such as E. coli. Constructs can be generated which exhibit anunexpected combination of properties. As shown in the Examples, aconstruct can be designed to be sufficiently hydrophilic to remainsoluble within the producing cell host, and fail to traverse theproducing host cell. The modified construct surprisingly retains the MTDfunction to traverse the bacterial outer cell wall to effect targetbacteria killing. This may be achieved because the bacterial cellmembrane properties (and structure) are sufficiently different from thebacterial outer membrane.

Such improved production constructs combine three properties: (1)produced in substantially soluble form in a host cell, typicallyGram-negative E. coli; (2) retains function of traversing the bacterialouter cell wall to access the periplasmic space where the substratepeptidoglycan is accessible to the catalytic domain; and (3) nosubstantial disruption of the inner membrane of the producing cell.Appropriate controls (such as the non-modified construct) will beincorporated to ensure that cell survival, expression, and catalyticactivity are be quantitated or relatively assessed.

Hydrophilicity positively affects protein solubility, and a protein withregions of concentrated hydrophobicity can be made more soluble bydisrupting such regions. As the MTD segments will typically be among themost hydrophobic segments of a chimeric construct, the MTD is mostamenable to amino acid substitution.

With certain insoluble (or minimally soluble) constructs from thesechimeras, the MTD segment is a short transmembrane segment. Knownhydrophobicity measurements can be used to locate transmembranesegments, which typically span about 20 amino acid residues. Decreasingthe overall hydrophobicity of these regions will often change theoverall protein solubility.

Regions of high hydrophobicity can be identified using DAS TMD analysis(see, e.g., Cserzo, et al. (1997) Protein Engineering 10(6):673-676),transmembrane using hidden Markov models (TMHMM) analysis (see, e.g.,Krogh, et al. (2001) J. Mol. Biol. 305(3):567-580), generalhydrophobicity (see, e.g., Kyte and Doolittle (1982) J. Mol. Biol.157(1):105-132), or the Grand Average of Hydropathy Score (GRAVY; seeGasteiger, et al (2005) “Protein Identification and Analysis Tools onthe ExPASy Server” in Walker (ed. 2005) The Proteomics ProtocolsHandbook, Humana Press, pp. 571-607).

The Dense Alignment Surface (DAS) prediction server is meant forpredicting transmembrane helices in membrane proteins. The program usesthe condition that membrane proteins are composed of stretches of 15-30predominantly hydrophobic residues separated by polar connecting loops.This means that the transmembrane region will detect a fragment that ispredominately composed of hydrophobic amino acids, flanked by residuesthat are hydrophilic or polar residues. DAS is based on low-stringencydot-plots of the query sequence against a collection of non-homologousmembrane proteins using a previously derived, special scoring matrix.Since integral membrane proteins are composed of more hydrophobicresidues than water soluble globular proteins, they can be discriminatedaccording to their composition. The principal difference between the DASmethod and the hyrdophobicity profile based programs is that DASdescribes the hydrophobic segments at three levels. This complexapproach of hydrophobicity is the key behind the sensitivity of the DASmethod.

DAS plots indicate a “strict” cutoff at 2.2 DAS score, and a “loose”cutoff at 1.7. The hit at 2.2 is informative in terms of the number ofmatching segments, while a hit at 1.7 gives the actual location of thetransmembrane segment. Typically, a DAS score of less than 3 (e.g., lessthan 2.8, 2.5, or 2.2) over the span of the protein indicates that aprotein will be soluble upon overexpression in a host cell.

Amino acids with electrically charged side chains include Arg, His, Lys(positively charged), with hydropathy scores of −4.5, −3.2, and −3.9respectively, and Glu and Asp (negatively charged), both with hydropathyscores of −3.5. Amino acids with polar but uncharged side chains includeSer, Thr, Asn, and Gin, with hydropathy scores of −0.8, −0.7, −3.5, and−3.2, respectively. Amino acids with non-polar (hydrophobic sidechains): Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val, with hydropathyscore being 1.8, 4.5, 3.8, 1.9, 2.8, −0.9, −1.3, and 4.2. Examples ofamino acids to substitute for valine include tyrosine, tryptophan,arginine, histidine, or lysine. Examples of amino acids to substitutefor isoleucine include tyrosine or tryptophan, arginine, histidine, orlysine. Examples of amino acids to substitute for leucine includetyrosine, tryptophan, arginine, histidine, or lysine.

For example, in FIG. 1A, the peak measure is above about 3.5 for theC-terminal MTD region. The segment was modified to decrease the localDAS profile score, which is reflected in FIG. 1B. Typically, substantialpeaks are targeted (e.g., those higher than about 3.1, 3.0, 2.9, 2.7,2.5) to lower local peak values to less than about 2.2, 2.1, 2.0, 1.8 or1.5. The decrease in DAS profile score will typically be at least about0.2, 0.3, 0.4, or 0.5 DAS units. The DAS profile of a MTD segment,however, must remain high enough to effectively transport a chimericprotein comprising the MTD across the outer membrane of a gram negativebacteria.

TMHMM is a software analysis based on a hidden Markov model. It predictstransmembrane helices and discriminates between soluble and membraneproteins with a high degree of accuracy. Methods for prediction oftransmembrane helices using hydrophobicity analysis alone are notreliable always. This method implicitly combines the hydrophobic signalto detect transmembrane (TM) segments and the charge bias, an abundanceof positively charged residues in the part of the sequence on thecytoplasmic side of the membrane protein into one integrated algorithm.Helical membrane proteins have alternating cytoplamic andnon-cytoplasmic loops. TMHMM can incorporate hydrophobicity, chargebias, helix lengths, and grammatical constraints into one model forprediction. This program allows one to predict the location oftransmembrane alpha helices and the location of intervening loop regionstogether with prediction of which loops between the helices will be onthe inside or outside of the cell or organelle. This program does notdetect beta sheet transmembrane domains. It takes about 20 amino acidsto span a lipid bilayer in an alpha helix. Programs can detect thesetransmembrane domains by looking for the presence of an alpha helix atleast about 20 amino acids long which contains hydrophobic amino acids.It correctly predicts 97-98% of the transmembrane helices while DenseAlignment Surface method (DAS) to predict transmembrane segments in anyintegral membrane protein. DAS has two levels of stringency which ismore comprehensive than TMHMM.

To improve solubility of a protein comprising a TMHMM predictedtransmembrane domain, hydrophobic amino acids can be substituted ormodified, e.g., to decrease the probability of a TMD by about 0.5, 0.6,0.7, 0.8, or even about 0.9 down to lower values, e.g., in the range ofabout 0.6 or lower ranges, with a reduction of about 0.2, 0.3, 0.4, or0.5.

A Kyte-Doolittle hydropathy plot is another predictive measure ofprotein structure, indicating transmembrane or surface regions. Thisdoes not predict secondary structure, so it will detect both alpha helixand beta sheet transmembrane domains. Numbers greater than 0 indicateincreased hydrophobicity, numbers less than 0 indicate an increase inhydrophilic amino acids.

First, each amino acid is given a hydrophobicity score between 4.6 and-4.6. A score of 4.6 is the most hydrophobic and a score of −4.6 is themost hydrophilic. After a window size is set, it is the number of aminoacids whose hydrophobicity scores will be averaged and assigned to thefirst amino acid in the window. The default window size is 9 aminoacids. The computer program starts with the first window of amino acidsand calculates the average of all the hydrophobicity scores in thatwindow. Then the computer program moves down one amino acid andcalculates the average of all the hydrophobicity scores in the secondwindow. This pattern continues to the end of the protein, computing theaverage score for each window and assigning it to the first amino acidin the window. The averages are then plotted on a graph. The y axisrepresents the hydrophobicity scores and the x axis represents thewindow number.

The Kyte-Doolittle scale is widely used for detecting hydrophobicregions in proteins. Regions with a positive value are hydrophobic,negative values are more hydrophilic. This scale can be used foridentifying both surface-exposed regions as well as transmembraneregions, depending on the used window size. Short window sizes of 5-7generally work well for predicting putative surface-exposed regions.Large window sizes of 19-21 are well suited for finding transmembranedomains if the values calculated are above about 1.6.

Typically, a Kyte-Doolittle score of less than 3 (e.g., less than 2.8,2.5, 2.2, or 2.0) over the span of the protein indicates that a proteinwill be soluble upon overexpression in a host cell. These values shouldbe used as a rule of thumb and deviations from the rule may occur.

Kyte and Doolittle also described an overall GRAVY score, which is theaverage hydropathy score for all the amino acids in the protein.Integral membrane proteins typically have higher GRAVY scores than doglobular proteins. This index is the general average hydropathicity(GRAVY) score for the hypothetical translated gene product. It iscalculated as the arithmetic mean of the sum of the hydropathic indicesof each amino acid.

Software to calculate GRAVY score is available free online on expasyProtparam. The input is the amino acid primary sequence in single letterformat. Since the score is an average value the parameter to be selectedis the window size to adjust the number of amino acids that are averagedto obtain an individual hydropathy score.

Typically, proteins with a negative GRAVY score are soluble (though suchproteins may associate structurally and functionally withmembrane-anchored proteins). Also, several hydrphilic proteins areretained in the lipophilic membrane fraction due to interaction withhydrophobic proteins (Althage, et al. (2004) Biochim Biophys Acta1659:73-82.; Guenebaut, et al. (1997) J. Mol. Biol. 265:409-418; andGuenebaut, et al. (1998) J. Mol. Biol. 276:105-112). GRAVY simplycalculates overall hydrophobicity of the linear polypeptide sequencewith increasing positive score indicating greater hydrophobicity, butdoes not account for order of residues, the way the protein folds inthree dimensions, or the percentage of residues buried in thehydrophobic core of the protein.

While not the most indicative measure, e.g., compared to DAS, a GRAVYscore of less than 1.5 (e.g., less than 1.2, 1.0, or 0.8) typicallyindicates that a protein will be soluble when expressed in a host cell.In the context of the presently described MTDs, the GRAVY score for theMTD should not dip below −1, to ensure the MTD retains outer membranetransversing activity.

For longer transmembrane or hydrophobic segments, one can localizehighly hydrophobic segments or amino acids to target for substitutionusing alternative methods. For example, one can determine surfaceexposed amino acid residues and their hydrophobicity index. If thehydrophobicity index or the GRAVY score is on the negative side, thenhydrophilic residues can be used to substitute the less hydrophilicmoieties. For example, the accessible surface area (ASA) orsolvent-accessible surface can be determined. ASA is the surface area ofa biomolecule that is accessible to a solvent (Lee and Richards (1971)J. Mol. Biol. 55:379-400). Solvent exposure of amino acids measures howdeep residues are buried in tertiary structure of proteins, and can beuseful for analyzing and predicting protein structure and function (Li,et al. (2011) Proteomics 11:3793-801; Ahmad, et al. (2003) Proteins50:629-35).

One can also use neural networks for prediction of surface exposedresidues. Data from protein crystal structures are used to teachcomputer-simulated neural networks rules for predicting surface exposurefrom sequence. These trained networks are able to correctly predictsurface exposure. See, e.g., Holbrook, et al. (1990) Protein Eng.3:659-665; Rost and Sander (1994) Proteins 20:216-226; Lebeda, et al.(1998) J. Protein Chem. 17:311-318; Pollastri, et al. (2002) Proteins47:142-153; and Ahmad and Gromiha (2002) Bioinformatics 18:819-824.Other approaches include logistic function (Mucchielli-Giorgi, et al.(1999 Bioinformatics 15:176-177); Bayersian analysis (Mucchielli-Giorgi,et al. (1999) Bioinformatics 15:176-177); Information theory(Naderi-Manesh, et al. (2001) Proteins 42:452-459; Richardson and Barlow(1999) Protein Eng. 12:1051-1054; and Carugo (2000) Protein Eng.13:607-609); and substitution matrices (Pascarella, et al. (1998)Proteins 32:190-199). A less quantitative approach to predict solventaccessibility is simply based on hydrophobicity plots (see Lesk (2002)Introduction to Bioinformatics Oxford University Press).

These methods indicate accessibility of each residue (exposed, buried,and, possibly, intermediate). Residues which are exposed to the solventare more likely to affect solubility and interact with aqueous solution.The exposed residues can be substituted with a more polar or hydrophilicresidue to improve solubility of the protein or domain upon expression.

The following references provide further guidance for improving proteinsolubility: Trevino, et al. (2007) J. Mol. Biol. 366:449-460; Magnan, etal. (2009) Bioinformatics 25:2200-2207; Ahuja, et al. (2006) MalariaJournal 5:52; Smialowski, et al. (2007) Bioinformatics 23:2536-2542;Trevino, et al. (2008) J. Pharm Sci 97: 4155-66; Makrides (1996)Microbiological Reviews 60:512-538; Karlsson, et al. (2005) J. Biol.Chem. 280:25558-564; Maxwell, et al. (1999) Protein Sci. 8:1908-1911;and Wilkinson and Harrison (1991) BioTechnology 9:443448.

X. Modification of Protein for Improved Half-Life, Stability, Storage

Addition of a polyalkylene glycol (PAG) moiety to a polypeptide or othermolecule is referred to as PAGylation. PAGylation (typically PEGylation)can improve the pharmacokinetic and pharmacodynamic properties of apolypeptide, e.g., increase in vitro and in vivo activity and stability,increase half-life, reduce proteolytic degradation, reduceimmunogenicity, and improve bioavailability and biodistribution. Aminoacids that are commonly targeted for PAGylation are arginine, asparticacid, glutamic acid, tyrosine, serine, threonine, histidine, cysteine,and lysine. The ε-amino group on the side chain of lysine is commonlytargeted for PAGylation.

Polyethylene glycol (PEG) is commercially available over a wide range ofmolecular weights from 300 g/mol to 10,000,000 g/mol. Different forms ofPEG can be made using different initiators for the polymerizationprocess, the most common of which is a monofunctional methyl ether PEG(methoxypoly(ethylene glycol)), referred to as mPEG.Lower-molecular-weight PEGs are also available as purer oligomers,referred to as monodisperse, uniform or discrete.

PEG molecules can have different geometries. Branched PEGs have three toten PEG chains emanating from a central core group. Star PEGs have10-100 PEG chains emanating from a central core group. Comb PEGs havemultiple PEG chains normally grafted to a polymer backbone.

Melting points vary depending on the MW of the polymer. PEG has thestructure:

HO—CH₂—(CH₂—O—CH₂—)_(n)—CH₂—OH

where n=9 would have an average molecular weight of approximately 400daltons (PEG 400). Ideally, for therapeutic consistency and regulatoryreasons, the PEG polymers added to the polypeptide are highly uniform(low dispersity). For a MW of about 5 KDa then n is about 100, and forthe MW of about 20 KDa n is about 400.

In addition to amino acid attachment site, considerations for thePEGylation reaction include the initiator PEGylating reagents, thePEG-to-protein ratio, pH, reaction time, and temperature (see, e.g.,Seely et al. (2005) “Making Site-specific PEGylation Work: Purificationand analysis of PEGylated protein pharmaceuticals presents manychallenges” BioPharm International).

Cys specific reagents include iodoacetamide or chloroacetamidechemistries, and maleimide chemistry has also been applied (Kalia andRaines (2010) Curr Org Chem. 14:138-147). PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone are thus useful reagentswhich allow cysteine specific PEGylation under mild conditions.

Agents that add PEG to the N-terminal amino acid of a given polypeptideinclude PEG NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate,and several others (Nucci et al. (1991) Adv. Drug Del. Rev. 6:133-151;Harris, et al. (1984) J. Poly. Sci: Polymer Chem. Ed. 22:341-352; Bailonand Berthold (1998) Pharm. Sci. Technol. Today 1:352-356). All reactunder mild conditions and are quite specific for amino groups. Generallythe pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus is attained.

PEG molecules and conditions for PEGylation are known in the art. See,e.g., Abuchowski, et al. (1984) Cancer Biochem. Biophys. 7:175-186;Abuchowski, et al. (1977) J. Biol. Chem. 252:3582-3586; Jackson. et al.(1987) Anal. Biochem. 165:114-127; Koide, et al. (1983) Biochem.Biophys. Res. Commun. 111:659-667; tresylate (Nilsson, et al. (1984)Methods Enzymol. 104:56-69; Delgado, et al. (1990) Biotechnol. Appl.Biochem. 12:119-128); N-hydroxysuccinimide derived active esters(Buckmann, et al. (1981) Makromol. Chem. 182:1379-1384; Joppich, et al.(1979) Makromol. Chem. 180:1381-1384; Abuchowski, et al. (1984) CancerBiochem. Biophys. 7:175-186; Katre, et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 84:1487-1491; Kitamura, et al. (1991) Cancer Res. 51:4310-4315;Boccu, et al. (1983) Z. Naturforsch. 38C:94-99); carbonates (Zalipsky,et al. pp. 347-370 in Harris (ed. 1992) Poly(ethylene glycol) Chemistry:Biotechnical and Biomedical Applications Plenum Press, New York;Zalipsky, et al. (1992) Biotechnol. Appl. Biochem. 15:100-114; Veronese,et al. (1985) Appl. Biochem. Biotech. 11:141-152); imidazolyl formates(Beauchamp, et al. (1983) Anal. Biochem. 131:25-33; Berger, et al.(1988) Blood 71:1641-1647); 4-dithiopyridines (Woghiren, et al. (1993)Bioconjugate Chem. 4:314-318); isocyanates (Byun, et al. (1992) ASAIOJournal M649-M653); and epoxides (U.S. Pat. No. 4,806,595, issued toNoishiki, et al. (1989)). Other linking groups include the urethanelinkage between amino groups and activated PEG (Veronese et al. (1985)Appl. Biochem. Biotechnol. 11:141-152.

A bifunctional PEG can be used to as a linker for the muralytic domainand MTD of the chimeric proteins described herein. For example,homobifunctional PEG can be used to conjugate the N-terminus of the MTDto the N-terminus of the catalytic domain (or vice versa). In someembodiments, a Y structure PEG derivative with one branch of the Yhaving an N-terminus specific group (e.g., aldehyde reactive group) andthe other with a C-terminus specific group (e.g., hydrazine) is used. Insome embodiments, a linear PEG with the reactive groups at either end isused.

In some embodiments, a cysteine residue is introduced on to the N or Cterminus of the domains, and a heterobifunctional PEG (e.g.,Thiol-PEG-Amine, a product with one end as thiol and the other end asamine) is used. Boc or Fmoc can be used to block amine groups.

Conjugation reactions can be performed in succession, and may involvepurification steps to remove undesired reactants and products. Thepurification methods will generally be typical peptide purificationmethods, many of which are known in protein chemistry. These may includesize exclusion chromatography, ion exchange chromatography, etc.

The PEG reacting group for a C terminus can be a hydrazine or similarspecific reacting group. The reaction will typically be for 0.5-18 hr;at an appropriate reaction temperature, e.g., between 20-40° C.; withappropriate peptide concentrations, e.g., 0.5-3 mg/ml; with appropriatePEG reagent concentrations, e.g., about 1-10 fold excess of PEG toprotein target; and appropriate pH, e.g., pH 4-7. The reaction can beterminated, the reactants removed, and the desired PEGylated polypeptideisolated.

The reaction linking the domain-PEG reacting group to the N terminus ofthe other domain can be an aldehyde or similar specific reacting group.Again, the reaction can run for an appropriate time, at an appropriatereaction temperature, with appropriate peptide concentrations, and withappropriate reagent concentrations, and appropriate pH.

Methoxypolyethylene glycol tresylate (mPEG-tresylate MW 5 kDA) can beused for lysine specific PEGylation of the target protein. Typically,mPEG-tresylate is incubated with the protein at 30° C. for 3 hr.

Methoxypolyethylene glycol maleimide (mPEG-maleimide, MW 5 kDA), acysteine specific PEG targeting reagent, can be used to PEGylate thedesired protein. Typically, mPEG-maleimide is reacted with the targetprotein at 30° C. for 3 hr.

Methoxypolyethylene glycol propionaldehyde is an N terminal specific PEGtargeting reagent (mPEG-aldehyde, MW 20 kDA). Typically, mPEG-aldehydeis incubated with the desired protein at 30° C. for 3-4 hr.

Standard SDS-PAGE electrophoresis is routinely employed to monitor thePEGylation reaction and the products of the derivitization. AnomalousSDS-PAGE migration as compared to molecular weight markers typicallyresults from the non-linear nature of the PEGylated products. Inparticular, because the PEG provides different SDS binding compared toprotein, migration of standard proteinaceous molecular weight markersdoes not correlate with the migration of protein derivatized withdifferent integral numbers of PEG moieties. The stoichiometry of bindingof SDS to the PEG is different from linear protein, and the charge ratiois non-linear.

The extent of PEGylation can be determined using a microfluidic basedelectrophoresis system, the Agilent 2100 Bioanalyzer (P230 assay). Forthe Bioanalyzer, protein loading and on-chip sample analysis wereperformed as described in the manufacturer's protocol. See Protein 230Kit Guide, Agilent Technologies Publication Number G2938-90054.PEGylation reactions typically result in differently PEGylated proteinspecies (un-, mono-, di-, tri-, etc.), having different numbers of PEGmoieties attached.

XI. Methodology

Production and use of the presently described chimeric polypeptidesinvolve well-known methods general clinical microbiology, generalmethods for handling bacteriophage, and general fundamentals ofbiotechnology, principles and methods. References for such methods arelisted below.

A. General Clinical Microbiology

General microbiology is the study of the microorganisms. See, e.g.,Sonenshein, et al. (ed. 2002) Bacillus Subtilis and Its ClosestRelatives: From Genes to Cells Amer. Soc. Microbiol.; Alexander andStrete (2001) Microbiology: A Photographic Atlas for the LaboratoryBenjamin/Cummings; Cann (2001) Principles of Molecular Virology (3ded.); Garrity (ed. 2005) Bergey's Manual of Systematic Bacteriology (2vol. 2d ed.) Plenum; Salyers and Whitt (2001) Bacterial Pathogenesis: AMolecular Approach (2d ed.) Amer. Soc. Microbiol.; Tierno (2001) TheSecret Life of Germs: Observations and Lessons from a Microbe HunterPocket Star; Block (ed. 2000) Disinfection Sterilization, andPreservation (5th ed.) Lippincott Williams & Wilkins Publ.; Cullimore(2000) Practical Atlas for Bacterial Identification Lewis Pub.; Madigan,et al. (2000) Brock Biology of Microorganisms (9th ed.) Prentice Hall;Maier, et al. (ads. 2000) Environmental Microbiology Academic Pr.;Tortora, et al. (2000) Microbiology: An Introduction includingMicrobiology Place™ Website, Student Tutorial CD-ROM, and Bacteria IDCD-ROM (7th ed.), Benjamin/Cummings; Demain, et al. (eds. 1999) Manualof Industrial Microbiology and Biotechnology (2d ed.) Amer. Soc.Microbiol.; Flint, et al. (eds. 1999) Principles of Virology: MolecularBiology, Pathogenesis, and Control Amer. Soc. Microbiol.; Murray, et al.(ed. 1999) Manual of Clinical Microbiology (7th ed.) Amer. Soc.Microbiol.; Burlage, et al. (eds. 1998) Techniques in Microbial EcologyOxford Univ. Press; Forbes, et al. (1998) Bailey & Scott's DiagnosticMicrobiology (10th ed.) Mosby, Schaechter, et al. (ed. 1998) Mechanismsof Microbial Disease (3d ed.) Lippincott, Williams & Wilkins; Tomes(1998) The Gospel of Germs: Men, Women, and the Microbe in American LifeHarvard Univ. Pr.; Snyder and Champness (1997) Molecular Genetics ofBacteria Amer. Soc. Microbiol., ISBN: 1555811027; Karlen (1996) MAN ANDMICROBES: Disease and Plagues in History and Modern Times TouchstoneBooks; and Bergey (ed. 1994) Bergey's Manual of DeterminativeBacteriology (9th ed.) Lippincott, Williams & Wilkins.

B. General Methods for Handling Bacteriophage

General methods for handling bacteriophage are well known, see, e.g.,Snustad and Dean (2002) Genetics Experiments with Bacterial VirusesFreeman; O'Brien and Aitken (eds. 2002) Antibody Phage Display: Methodsand Protocols Humana; Ring and Blair (eds. 2000) Genetically EngineeredViruses BIOS Sci. Pub.; Adolf (ed. 1995) Methods in Molecular Genetics:Viral Gene Techniques vol. 6, Elsevier; Adolf (ed. 1995) Methods inMolecular Genetics: Viral Gene Techniques vol. 7, Elsevier; and Hobanand Rott (eds. 1988) Molec. Biol. of Bacterial Virus Systems (CurrentTopics in Microbiology and Immunology No. 136) Springer-Verlag.

C. General Fundamentals of Biotechnology, Principles and Methods

General fundamentals of biotechnology, principles and methods aredescribed, e.g., in Alberts, et al. (2002) Molecular Biology of the Cell(4th ed.) Garland; Lodish, et al. (1999) Molecular Cell Biology (4thed.) Freeman; Janeway, et al. (eds. 2001) Immunobiology (5th ed.)Garland; Flint, et al. (eds. 1999) Principles of Virology: MolecularBiology, Pathogenesis, and Control, Am. Soc. Microbiol.; Nelson, et al.(2000) Lehninger Principles of Biochemistry (3d ed.) Worth; Freshney(2000) Culture of Animal Cells: A Manual of Basic Technique (4th ed.)Wiley-Liss; Arias and Stewart (2002) Molecular Principles of AnimalDevelopment, Oxford University Press; Griffiths, et al. (2000) AnIntroduction to Genetic Analysis (7th ed.) Freeman; Kierszenbaum (2001)Histology and Cell Biology, Mosby; Weaver (2001) Molecular Biology (2ded.) McGraw-Hill; Barker (1998) At the Bench: A Laboratory Navigator CSHLaboratory; Branden and Tooze (1999) Introduction to Protein Structure(2d ed.), Garland Publishing; Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual (3 vol., 3d ed.), CSH Lab. Press; Scopes(1994) Protein Purification: Principles and Practice (3d ed.) SpringerVerlag; Simpson, et al. (eds. 2009) Basic Methods in ProteinPurification and Analysis: A Laboratory Manual, CSHL Press, NY, ISBN978-087969868-3; Friedmann and Rossi (eds. 2007) Gene Transfer: Deliveryand Expression of DNA and RNA, A Laboratory Manual, CSHL Press, NY, ISBN978-087969764-8; Link and LaBaer (2009) Proteomics: A Cold Spring HarborLaboratory Course Manual, CSHL Press, NY, ISBN 978-087969793-8; andSimpson (2003) Proteins and Proteomics: A Laboratory Manual, CSHL Press,NY, ISBN 978-087969554-5, Other references directed to bioinformaticsinclude, e.g., Mount (2004) Bioinformatics: Sequence and Genome Analysis(2d ed.), CSHL Press, NY, ISBN 978-087969687-0; Pevsner (2009)Bioinformatics and Functional Genomics (2d ed.) Wiley-Blackwell,ISBN-10: 0470085851, ISBN-13: 978-0470085851; Lesk (2008) Introductionto Bioinformatics (3d ed.) Oxford Univ. Press, ISBN-10: 9780199208043,ISBN-13: 978-0199208043; Zvelebil and Baum (2007) UnderstandingBioinformatics, Garland Science, ISBN-10: 0815340249, ISBN-13:978-0815340249; Baxevanis and Ouellette (eds. 2004) Bioinformatics: APractical Guide to the Analysis of Genes and Proteins (3d ed.)Wiley-Interscience; ISBN-10: 0471478784, ISBN-13: 978-0471478782; Gu andBourne (eds. 2009) Structural Bioinformatics (2d ed.), Wiley-Blackwell,ISBN-10: 0470181052, ISBN-13: 978-0470181058; Selzer, et al. (2008)Applied Bioinformatics: An Introduction, Springer, ISBN-10:9783540727996, ISBN-13: 978-3540727996; Campbell and Heyer (2006)Discovering Genomics, Proteomics and Bioinformatics (2d ed.), BenjaminCummings, ISBN-10: 9780805382198, ISBN-13: 978-0805382198; Jin Xiong(2006) Essential Bioinformatics, Cambridge Univ Press, ISBN-10:0521600820, ISBN-13: 978-0521600828; Krane and Raymer (2002) FundamentalConcepts of Bioinformatics, Benjamin Cummings, ISBN-10: 9780805346336,ISBN-13: 978-0805346336; He and Petoukhov (2011) Mathematics ofBioinformatics: Theory, Methods and Applications (Wiley Series inBioinformatics), Wiley-Interscience, ISBN-10: 9780470404430, ISBN-13:978-0470404430; Altemvitz and Ramoni (2011) Knowledge-BasedBioinformatics: From analysis to interpretation, Wiley, ISBN-10:9780470748312, ISBN-13: 978-0470748312; Gopakumar (2011) Bioinformatics:Sequence and Structural Analysis, Alpha Science Intl Ltd., ISBN-10:184265490X, ISBN-13: 978-1842654903; Barnes (ed. 2007) Bioinformaticsfor Geneticists: A Bioinformatics Primer for the Analysis of GeneticData (2d ed.) Wiley, ISBN-10: 9780470026199, ISBN-13: 978-0470026199;Neapolitan (2007) Probabilistic Methods for Bioinformatics, KaufmannPublishers, ISBN-10: 0123704766, ISBN-13: 978-0123704764; Rangwala andKarypis (2010) Introduction to Protein Structure Prediction: Methods andAlgorithms (Wiley Series in Bioinformatics), Wiley, ISBN-10: 0470470593,ISBN-13: 978-0470470596; Ussery, et al. (2010) Computing for ComparativeMicrobial Genomics: Bioinformatics for Microbiologists (ComputationalBiology), Springer, ISBN-10: 9781849967631, ISBN-13: 978-1849967631; andKeith (ed. 2008) Bioinformatics: Volume I: Data, Sequence Analysis andEvolution (Methods in Molecular Biology), Humana Press, ISBN-10:9781588297075, ISBN-13: 978-1588297075.

The following references provide additional guidance for fusion andchimeric proteins: Hammarstrom, et al. (2001) Protein Science11:313-321; Harrison (1999) InNovations 11:4-7; Banerjee and PadmanabhanWO/2010/125588.

D. Mutagenesis; Site Specific, Random, Shuffling

Based upon the structural and functional descriptions provide herein,homologs and functional variants can be generated. Segments withpenetration functions can be found by structural homology. Phage tailgenes are typically found in particular gene arrangements, and otherentities found in the corresponding arrangements can be tested for cellwall degrading function. These may also serve as the starting points toscreen for variants of the structures, e.g., mutagenizing suchstructures and screening for those which have desired characteristics,e.g., broader substrate specificity. Standard methods of mutagenesis maybe used, see. e.g., Johnson-Boaz, et al. (1994) Mol. Microbiol.13:495-504; U.S. Pat. Nos. 6,506,602, 6,518,065, 6,521,453, 6,579,678.

Membrane transfer segments can be similarly identified, and prevalent orspecific target motifs can be screened for receptor domains whichspecifically interact. Targets can be surface expressed proteins,carbohydrate, or lipid containing structures found on the various targetstrains. Mutagenesis can be used to broaden binding selectivity orincrease stability of segments or the entire construct, deletionstrategies may eliminate extraneous segments.

The components of the Gram-positive bacteria cell wall can be sharedwith components of the Gram-negative cell wall, or with othermycobacteria or spores. Other phage derived activities can be combinedto penetrate more complex Gram-negative cell wall structures ifnecessary. In particular, multiple catalytic segments can be used toprovide multiple activities, which can function synergistically within asingle construct or when combined with another therapeutic, e.g.,antibiotic or antimicrobial.

A targeting moiety can increase a local concentration of an activemoiety, but a linker of appropriate length may also increase the numberof wall degrading events locally. Thus, linkers compatible with thetarget and cell wall degrading segment, or of appropriate length, can beused to increase the cell wall penetration activity leading to stasis orkilling of target bacteria.

Phage have been selected to survive outside of cells, often underbiologically inhospitable conditions. Thus, the structures are likely tobe particularly hardy and robust, and resistant to the environmentalconditions which might otherwise inactivate enzymatic or catalyticentities. Bacteria which live in inhospitable environments, e.g.,extreme environments of temperature, salt, oxidizing or reactiveextremes, high pressure, etc., are targeted by phage which areparticularly adapted to survive those conditions. Polypeptides derivedfrom these phage are likely to be more stable in various purificationprocesses, storage, and pharmacological conditions of use.

E. Screening

Screening methods can be devised for evaluating mutants or new candidatemuralytic segments. A purified preparation of phage particles can bescreened for presence of such gene products on the phage structure.

Muralytic activity screens can use crude bacteria cultures, isolatedbacterial cell wall components, peptidoglycan preparations, syntheticsubstrates, or purified reagents to determine the affinity and number ofsubstrate sites on target cells. Penetration or wall degrading assayscan be incorporated to evaluate integrity of the outer membranes oftarget strains, lawn inhibition assays, viability tests of cultures,activity on cell wall preparations or other substrates, or release ofcomponents (e.g., sugars, amino acids, polymers) of the cell wall uponmuralytic action. Amidase activity may be measured by release of solubleN-acetyl hexose amines (e.g., modified Morgan-Elson reaction) orendopeptidase activity by assay for free amino groups (L-alanine forala-gly endopeptidases, L-glycine for gly-gly endopeptidases) using aDNFB assay), all three of these assays based on Petit et al. (1966)Biochemistry 5:2764-76. Gly-gly endopeptidase activity can also bemeasured as the release of free amino groups from N-acetylatedhexaglycine (acetyl-Gly6), see Kline, et al. (1994) Anal. Biochem.217:329-331.

Linkers can be tested to compare the effects on membrane transfer ordegradation, or to compare the activities of various orientations of theactive fragments. Panels of targets (e.g., Gram-negative, Gram-positive,mycobacteria and spores) can be screened using cell wall degradingfragments to determine which fragments on a broader or narrower spectrumof targets.

One method to test for a cell wall degrading activity is to treat phagewith mild detergents or denaturants to release proteins associated withthe virion. These proteins are further tested for wall degrading ormuralytic activity on bacterial cells. Another method is to determinecell wall degradation activity or lysis from without (LO) on a phageresistant host. A third method to assess wall degrading or muralyticactivity associated with phage structural component is to performZymogram assays, e.g., where a pure phage preparation is electrophoresedon SDS-polyacrylamide gel incorporating autoclaved host cells. Proteinson the gels are allowed to renature in situ and then act upon the cellwall components giving rise to clear “muralytic” zones when the rest ofthe gel stains blue with methylene blue dye. See, e.g., Lepeuple, et al,(1998) Appl. Environ. Microbiol. 64:4142-428. The clear zones arevisualized and the protein band from each zone is eluted. The proteincan be identified, e.g., by N-terminal sequencing or by Massspectrometry. The coding sequence for the degrading protein can then beisolated.

XII. Isolation of Nucleic Acids Encoding MTDs and/or Muralytic Domains

Further provided are nucleic acids that encode the cell wall degradingor membrane transfer domains. Such polynucleotides encode muralyticproteins described herein, including proteins with CHAP domains(particularly C terminal CHAP domains) and others with cell walldegrading activity.

Nucleic acids that encode cell wall degrading polypeptides are relevantto the nucleic acid embodiments of the invention. These nucleic acids(e.g., cDNA, genomic, or subsequences (probes)) can be cloned, oramplified by in vitro methods such as the polymerase chain reaction(PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), or the self-sustained sequence replicationsystem (SSR). Besides synthetic methodologies, a wide variety of cloningand in vitro amplification methodologies are well-known to persons ofskill. Examples of these techniques and instructions sufficient todirect persons of skill through many cloning exercises are found inBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology 152 Academic Press, Inc.; Sambrook et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press; Current Protocols in MolecularBiology, Ausubel et al., eds., Current Protocols (Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., 1994 Supplement); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0246864.

A DNA that encodes a cell wall degrading polypeptide can be prepared bya suitable method described above, including, e.g., cloning andrestriction of appropriate sequences with restriction enzymes. Nucleicacids encoding cell wall degrading polypeptides can be isolated byroutine cloning methods. An exemplary nucleotide sequence of a cell walldegrading polypeptide, e.g., in Accession Number YP_024486, can be usedto design probes that specifically hybridize to a gene; or to an mRNA,encoding a cell wall degrading protein, in a total nucleic acid sample(e.g., in a Southern or Northern blot). Once the target nucleic acidencoding the cell wall degrading protein is identified, it can beisolated according to standard methods known to those of skill in theart. Further, the isolated nucleic acids can be cleaved with restrictionenzymes to create nucleic acids encoding the full-length cell walldegrading polypeptide, or subsequences thereof, e.g., containingsubsequences encoding at least a subsequence of a catalytic domain of acell wall degrading polypeptide. These restriction enzyme fragments,encoding a cell wall degrading polypeptide or subsequences thereof, canthen be ligated, for example, to produce a nucleic acid encoding a cellwall degrading polypeptide.

Similar methods can be used to generate appropriate cell wall bindingfragments or linkers between fragments.

A nucleic acid encoding an appropriate polypeptide, or a subsequencethereof, can be characterized by assaying for the expressed product.Assays based on the detection of the physical, chemical, orimmunological properties of the expressed polypeptide can be used. Forexample, one can identify a cell wall degrading polypeptide by theability of a polypeptide encoded by the nucleic acid to degrade ordigest bacterial cells, e.g., as described herein

Also, a nucleic acid encoding a desired polypeptide, or a subsequencethereof, can be chemically synthesized. Suitable methods include thephosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99;the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Terra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat.No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skillrecognizes that while chemical synthesis of DNA is often limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Nucleic acids encoding a desired polypeptide, or subsequences thereofcan be cloned using DNA amplification methods such as polymerase chainreaction (PCR). Thus, for example, the nucleic acid sequence orsubsequence is PCR amplified, using a sense primer containing onerestriction enzyme site (e.g., NdeI) and an antisense primer containinganother restriction enzyme site (e.g., HindIII). This will produce anucleic acid encoding the desired polypeptide or subsequence and havingterminal restriction enzyme sites. This nucleic acid can then be easilyligated into a vector containing a nucleic acid encoding the secondmolecule and having the appropriate corresponding restriction enzymesites. Suitable PCR primers can be determined by one of skill in the artusing the sequence information provided in GenBank or other sources.Appropriate restriction enzyme sites can also be added to the nucleicacid encoding the cell wall degrading polypeptide or a polypeptidesubsequence thereof by site-directed mutagenesis. The plasmid containinga cell wall degrading polypeptide-encoding nucleotide sequence orsubsequence is cleaved with the appropriate restriction endonuclease andthen ligated into an appropriate vector for amplification and/orexpression according to standard methods. Examples of techniquessufficient to direct persons of skill through in vitro amplificationmethods are found in Berger, Sambrook, and Ausubel, as well as Mullis etal. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al., eds) Academic Press Inc. (1990); Arnheim &Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al(1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace(1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117.

Some nucleic acids encoding cell wall degrading polypeptides can beamplified using PCR primers based on the sequence of the identifiedpolypeptides.

Other physical properties, e.g., of a recombinant cell wall degradingpolypeptide expressed from a particular nucleic acid, can be compared toproperties of known desired polypeptides to provide another method ofidentifying suitable sequences or domains, e.g., of the cell walldegrading proteins that are determinants of bacterial specificity,binding specificity, and/or catalytic activity. Alternatively, a cellwall degrading polypeptide encoding nucleic acid or recombinant cellwall degrading polypeptide gene can be mutated, and its role as a cellwall degrading polypeptide, or the role of particular sequences ordomains established by detecting a variation in bacterial “lysis”normally enhanced by the unmutated, naturally-occurring, or control cellwall degrading polypeptide. Those of skill will recognize that mutationor modification of cell wall degrading polypeptides of the invention canbe facilitated by molecular biology techniques to manipulate the nucleicacids encoding the polypeptides, e.g., PCR. Other mutagenesis or geneshuffling techniques may be applied to the functional fragmentsdescribed herein, including wall degrading activities, wall bindingproperties, or linker features compatible with chimeric constructs.

Functional domains of newly identified cell wall degrading polypeptidescan be identified by using standard methods for mutating or modifyingthe polypeptides and testing them for activities such as acceptorsubstrate activity and/or catalytic activity, as described herein. Thesequences of functional domains of the various cell wall degradingproteins can be used to construct nucleic acids encoding or combiningfunctional domains of one or more cell wall degrading polypeptides.These multiple activity polypeptide fusions can then be tested for adesired bacteriostatic or bacteriolytic activity. Particular examples ofsources for cell wall degrading polypeptides include prophage sequences,including incomplete remnants of functional phage genomes, orpyocin-like structures, including particles derived from phage-likegenetic segments, e.g., deletion or mutated genetic remnants of phageremaining in the DNA of a bacterium.

Nucleic acids encoding cell wall degrading polypeptides can beidentified by alignment and comparison with known nucleic acid or aminoacid sequences of cell wall degrading polypeptides, e.g., to determinethe amount of sequence identity between them. This information can beused to identify and select polypeptide domains that confer or modulatecell wall degrading polypeptide activities, e.g., target bacterial orbinding specificity and/or degrading activity based on the amount ofsequence identity between the polypeptides of interest. For example,domains having sequence identity between the cell wall degradingpolypeptides of interest, and that are associated with a known activity,can be used to construct polypeptides containing that domain and otherdomains, and having the activity associated with that domain (e.g.,bacterial or binding specificity and/or wall degrading activity).Similar strategies may be applied to isolate bacterial SH3 domains whichbind to cell wall structures, peptidoglycan recognizing proteins(PGRPs), phage tail “muralytic” polypeptides, or to linkers for spacingbetween domains.

XIII. Expression of Desired Polypeptides in Host Cells

The proteins described herein can be expressed in a variety of hostcells, including E. coli, other bacterial hosts, and yeast. The hostcells can be microorganisms, such as, for example, yeast cells,bacterial cells, or filamentous fungal cells. Examples of suitable hostcells include, for example, Azotobacter sp. (e.g., A. vinelandii),Pseudomonas sp., Rhizobium sp., Erwinia sp., Escherichia sp. (e.g., E.coli), Bacillus, Pseudomonas, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoascilla, Paracoccus, Staphylococcus, and Klebsiella sp.,among many others. The cells can be of any of several genera, includingSaccharomyces (e.g., S. cerevisiae), Candida (e.g., C. utilis, Cparapsilosis, C. krusei, C. versatllis, C. lipolytica, C. zeylanoides,C. guilliermondii, C. albicans, and C. humicola), Pichia (e.g., P.farinosa and P. ohmeri), Torulopsis (e.g., T. candida, T. sphaerica, T.xylinus, T. famata, and T. versatilis), Debaryomyces (e.g., D.subglobosus, D. cantarellii, D. globosus, D. hansenii, and D.japonicus), Zygosaccharomyces (e.g., Z. rouxii and Z. bailii),Kluyveromyces (e.g., K. marxianus), Hansenula (e.g., H. anomala and H.jadinii), and Brettanomyces (e.g., B. lambicus and B. anomalus).Examples of useful bacteria include, but are not limited to,Escherichia, Enterobacter, Azotobacter, Erwinia, Klebsielia, Bacillus,Pseudomonas, Proteus, and Salmonella. Eukaryotic cells, e.g., CHO cells,can also be used for production.

Once expressed in a host cell, the cell wall degrading polypeptides canbe used to prevent growth or kill target bacteria. In some embodiments,the P225 polypeptide (SEQ ID NO:9) is used to decrease growth of aGram-negative bacterium. In some embodiments, the protein is used todecrease growth of a Pseudomonas, e.g., Pseudomonas aeruginosa,bacterium. Fusion constructs combining such fragments can be generated,including fusion proteins comprising a plurality of wall degradingactivities, including both peptidase and amidase catalytic activities(which cleave both gly-gly and gly-ala linkages).

Typically, a polynucleotide that encodes the cell wall degradingpolypeptides is placed under the control of a promoter that isfunctional in the desired host cell. An extremely wide variety ofpromoters is well known, and can be used in expression vectors of theinvention, depending on the particular application. Ordinarily, thepromoter selected depends upon the cell in which the promoter is to beactive. Other expression control sequences such as ribosome bindingsites, transcription termination sites, etc., can be included.Constructs that include one or more of these control sequences aretermed “expression cassettes.” Accordingly, the invention providesexpression cassettes into which the nucleic acids that encode fusionproteins, e.g., combining a cell wall degrading fragment with an outermembrane binding fragment, are incorporated for expression in a desiredhost cell.

Expression control sequences that are suitable for use in a particularhost cell can be obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems, the tryptophan (trp) promoter system(Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the tac promoter(DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25); and thelambda-derived P_(L) promoter and N-gene ribosome binding site(Shimatake et al. (1981) Nature 292: 128). A bacteriophage T7 promoteris used in various examples, though one of skill will recognize that theparticular promoter system is not critical to the invention.

For expression of cell wall degrading polypeptides in prokaryotic cellsother than E. coli, a promoter that functions in the particularprokaryotic production species is used. Such promoters can be obtainedfrom genes that have been cloned from the species, or heterologouspromoters can be used. For example, the hybrid trp-lac promoterfunctions in Bacillus in addition to E. coli.

Hyper-expression of proteins can result in inclusion body formation. Thestronger the promoter, the higher is the protein yield per cell, and insome cases, a medium strength promoter results in higher yield ofsoluble protein compared to a strong promoter. Some examples include T7promoter, arabinose promoters, T5 and hybrid promoters, etc. Moreover,some toxic protein has been found to be difficult to express due toleaky expression in bacterial cells. Such leaky expression can beavoided by use of promoters that are strongly regulated like arabinosepromoters. See, e.g., Correa and Oppezzo (2011) Biotechnol. J. 6:715-730and Alakomi (2007) “Weakening of the Gram-negative bacterial outermembrane: A tool for increasing microbiological safety” thesis, UnivHelsinki, June 2007.

A ribosome binding site (RBS) is conveniently included in the expressioncassettes of the invention. An exemplary RBS in E. coli consists of anucleotide sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon (Shine and Dalgarno (1975) Nature254:34; Steitz (1979) In Biological regulation and development: Geneexpression (ed. R. F. Goldberger), vol. 1, p. 349, Plenum Publishing,NY).

For expression of proteins in yeast, convenient promoters includeGAL1-10 (Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2(Russell et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J.(1982) 6:675-680), and MFα (Herskowitz and Oshima (1982) in TheMolecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, andBroach) Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209).Another suitable promoter for use in yeast is the ADH2/GAPDH hybridpromoter as described in Cousens et al. (1987) Gene 61:265-275. Forfilamentous fungi such as, for example, strains of the fungi Aspergillus(McKnight et al., U.S. Pat. No. 4,935,349), examples of useful promotersinclude those derived from Aspergillus nidulans glycolytic genes, suchas the ADH3 promoter (McKnight et al., EMBO J. 4: 2093 2099 (1985)) andthe tpiA promoter. An example of a suitable terminator is the ADH3terminator (McKnight et al.).

Either constitutive or regulated promoters can be used in the presentinvention. Regulated promoters can be advantageous because the hostcells can be grown to high densities before expression of the fusionproteins is induced. High level expression of heterologous polypeptidesslows cell growth in some situations. An inducible promoter is apromoter that directs expression of a gene where the level of expressionis alterable by environmental or developmental factors such as, forexample, temperature, pH, anaerobic or aerobic conditions, light,transcription factors and chemicals. Such promoters are referred toherein as “inducible” promoters, which allow one to control the timingof expression of the desired polypeptide. For E. coli and otherbacterial host cells, inducible promoters are known to those of skill inthe art. These include, for example, the lac promoter, the bacteriophagelambda P_(L) promoter, the hybrid trp-lac promoter (Amann et al. (1983)Gene 25: 167; de Boer et al. (1983) Proc. Nat'l. Acad. Sci. USA 80: 21),and the bacteriophage T7 promoter (Studier et al. (1986) J. Mol. Biol.;Tabor et al. (1985) Proc. Nat'l. Acad. Sci. USA 82: 1074-8). Thesepromoters and their use are discussed in Sambrook et al., supra.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. A plethora of kits arecommercially available for the purification of plasmids from bacteria(see, e.g., EasyPrepJ, FlexiPrepJ, both from Pharmacia Biotech;StrataCleanJ, from Stratagene; and, QIAexpress Expression System,Qiagen). The isolated and purified plasmids can then be furthermanipulated to produce other plasmids, and used to transfect cells.Cloning in Streptomyces or Bacillus is also possible.

Selectable markers are often incorporated into the expression vectorsused to express the polynucleotides of the invention. These genes canencode a gene product, such as a polypeptide, necessary for the survivalor growth of transformed host cells grown in a selective culture medium.A number of selectable markers are known to those of skill in the artand are described for instance in Sambrook et al., supra.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques as described inthe references cited above. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required. To confirm correct sequences in plasmids constructed,the plasmids can be analyzed by standard techniques such as byrestriction endonuclease digestion, and/or sequencing according to knownmethods. Molecular cloning techniques to achieve these ends are known inthe art. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant nucleic acids arewell-known to persons of skill.

A variety of common vectors suitable for use as starting materials forconstructing the expression vectors of the invention are well known inthe art. For cloning in bacteria, common vectors include pBR322 derivedvectors such as pBLUESCRIP™, and X-phage derived vectors. In yeast,vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expression inmammalian cells can be achieved using a variety of commonly availableplasmids, including pSV2, pBC12BI, and p91023, as well as lytic virusvectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomalvirus vectors (e.g., bovine papillomavirus), and retroviral vectors(e.g., murine retroviruses).

Expression vectors can be introduced into a chosen host cell usingstandard methods known to those of skill in the art. For example, theexpression vectors can be introduced into prokaryotic cells, includingE. coli, by calcium chloride transformation, and into eukaryotic cellsby calcium phosphate treatment or electroporation.

Translational coupling can be used to enhance expression. The strategyuses a short upstream open reading frame derived from a highly expressedgene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few aminoacid codons by a termination codon. Just prior to the termination codonis a second ribosome binding site, and following the termination codonis a start codon for the initiation of translation. The system dissolvessecondary structure in the RNA, allowing for the efficient initiation oftranslation. See Squires, et. al. (1988), J. Biol. Chem. 263:16297-16302.

The various polypeptides of the invention can be expressedintracellularly, or can be secreted from the cell. Intracellularexpression often results in high yields. If necessary, the amount ofsoluble, active fusion polypeptide may be increased by performingrefolding procedures (see, e.g., Sambrook et al., supra.; Marston et al.(1984) Bio/Technology 2:800; Schoner et al. (1985) Bio/Technology3:151). In embodiments in which the polypeptide is secreted, either intothe periplasm or into the extracellular medium, the DNA sequence isoften linked to a cleavable signal peptide sequence. The signal sequencedirects translocation of the fusion polypeptide through the cellmembrane. An example of a suitable vector for use in E. coli thatcontains a promoter-signal sequence unit is pTA1529, which has the E.coli phoA promoter and signal sequence (see, e.g., Sambrook et al.,supra.; Oka et al. (1985) Proc. Nat. Acad. Sci. USA 82:7212; Talmadge etal. (1980) Proc. Natl. Acad. Sci. USA 77:3988; Takahara et al. (1985) J.Biol. Chem. 260:2670). In another embodiment, the fusion polypeptidesare fused to a subsequence of protein A or bovine serum albumin (BSA),for example, to facilitate purification, secretion, or stability.Affinity methods, e.g., using substrate for the catalytic fragment maybe appropriate.

The cell wall degrading polypeptides of the invention can also befurther linked to other polypeptide segments, e.g., biofilm depolymerasesegments. This approach often results in high yields, because normalprokaryotic control sequences direct transcription and translation. InE. coli, lacZ fusions are often used to express heterologous proteins.Suitable vectors are readily available, such as the pUR, pEX, and pMR100series. For certain applications, it may be desirable to cleaveextraneous sequence from the fusion polypeptide after purification. Thiscan be accomplished by any of several methods known in the art,including cleavage by cyanogen bromide, a protease, or by Factor X_(a)(see, e.g., Sambrook et al., supra.; Itakura et al. (1977) Science198:1056; Goeddel et al. (1979) Proc. Natl. Acad. Sci. USA 76:106; Nagaiet al. (1984) Nature 309:810; Sung et al. (1986) Proc. Natl. Acad. Sci.USA 83:561). Cleavage sites can be engineered into the gene for thefusion polypeptide at the desired point of cleavage.

More than one recombinant polypeptide may be expressed in a single hostcell by placing multiple transcriptional cassettes in a singleexpression vector, or by utilizing different selectable markers for eachof the expression vectors which are employed in the cloning strategy.

A suitable system for obtaining recombinant proteins from E. coli whichmaintains the integrity of their N-termini has been described by Milleret al (1989) Biotechnology 7:698-704. In this system, the gene ofinterest is produced as a C-terminal fusion to the first 76 residues ofthe yeast ubiquitin gene containing a peptidase cleavage site. Cleavageat the junction of the two moieties results in production of a proteinhaving an intact authentic N-terminal reside.

XIV. Purification of Desired Polypeptides

A crude cellular extract containing the expressed intracellular orsecreted polypeptides described herein can be used in the methods of thepresent invention.

The polypeptides can also be purified according to standard proceduresof the art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like (see, generally,R. Scopes (1982) Protein Purification, Springer-Verlag, N.Y., Deutscher(1990) Methods in Enzymology Vol. 182: Guide to Protein Purification,Academic Press, Inc. N.Y.). Because the degrading segments, at least,derive from phage proteins selected for stability, purification caninvolve denaturation of contaminating materials. Substantially purecompositions are typically about 70, 75, 80, 85, 90, 92, 95, 98 to 99%or higher homogeneous. The purified polypeptides can also be used, e.g.,as immunogens for antibody production, which antibodies may be used inimmunoselection purification methods.

To facilitate purification of the polypeptides of the invention, thenucleic acids that encode them can also include a coding sequence for anepitope or “tag” for which an affinity binding reagent is available,e.g., a purification tag. Examples of suitable epitopes include the mycand V-5 reporter genes: expression vectors useful for recombinantproduction of fusion polypeptides having these epitopes are commerciallyavailable (e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-Hisand pcDNA3.1/VS-His are suitable for expression in mammalian cells).Additional expression vectors suitable for attaching a tag to thepolypeptides of the invention, and corresponding detection systems areknown to those of skill in the art, and several are commerciallyavailable (e.g., FLAG, Kodak, Rochester N.Y.). Another example of asuitable tag is a polyhistidine sequence, which is capable of binding tometal chelate affinity ligands. Typically, six adjacent histidines areused, although one can use more or fewer than six. Suitable metalchelate affinity ligands that can serve as the binding moiety for apolyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli (1990)Genetic Engineering: Principles and Methods, J. K. Setlow, Ed., PlenumPress, NY; commercially available from Qiagen (Santa Clarita, Calif.)).Purification tags also include maltose binding domains and starchbinding domains. Purification of maltose binding domain proteins isknown to those of skill in the art.

Other haptens that are suitable for use as tags are known to those ofskill in the art and are described, for example, in the Handbook ofFluorescent Probes and Research Chemicals (6th Ed., Molecular Probes,Inc., Eugene Oreg.). For example, dinitrophenol (DNP), digoxigenin,barbiturates (see, e.g., U.S. Pat. No. 5,414,085), and several types offluorophores are useful as haptens, as are derivatives of thesecompounds. Kits are commercially available for linking haptens and othermoieties to proteins and other molecules. For example, where the haptenincludes a thiol, a heterobifunctional linker such as SMCC can be usedto attach the tag to lysine residues present on the capture reagent.

One of skill will recognize that certain modifications can be made tothe catalytic or functional domains of the polypeptide withoutdiminishing their biological activity. Some modifications can be made tofacilitate the cloning, expression, or incorporation of the catalyticdomain into a fusion polypeptide. Such modifications are well known tothose of skill in the art and include, for example, the addition ofcodons at either terminus of the polynucleotide that encodes thecatalytic domain, e.g., a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionenzyme sites or termination codons or purification sequences.

The following discussion of the invention is for the purposes ofillustration and description, and is not intended to limit the inventionto the form or forms disclosed herein. Although the description of theinvention has included description of one or more embodiments andcertain variations and modifications, other variations and modificationsare within the scope of the invention, e.g., as may be within the skilland knowledge of those in the art, after understanding the presentdisclosure. All publications, patents, patent applications, Genbanknumbers, and websites cited herein are hereby incorporated by referencein their entireties for all purposes.

XV. EXAMPLES Example 1. GP36 Enzymatic Test Construct A. MuralyticDomain Construct

We isolated the ORF36 sequence from Pseudomonas phage P134 (SEQ ID NO:1). The translation product is shown as SEQ ID NO:2. Based on analysisof the sequence, a murein degrading catalytic domain fragment wasidentified spanning amino acids 737-875 of SEQ ID NO:2. An expressionvector was constructed with a promoter and adjacent initiation codonlinked to amino acid 683, so that the nucleic acid construct encodesamino acids 683-898. See SEQ ID NO: 5, which is translated as SEQ ID NO:6. This segment was designated the GP36 CD fragment.

A sequence was added (thereby removing the termination codon from theend of GP36) to provide an extension of 13 amino acids comprising a 6His purification tag (for purification on a Nickel column). Theconstruct results in a polypeptide having SEQ ID NO: 7.

The expression construct was introduced into E. coli, production ofprotein was induced, and cells harvested. The cell pellets wereprocessed to isolate the protein, and purified using affinitychromatography over Ni-NTA resin using standard techniques. The proteinwas analyzed to be greater than 95% pure by SDS-PAGE.

B. Bacterial Killing Assay

As indicated above, the bacterial outer membrane in a Gram-negativebacteria prevents access to the peptidoglycan from the externalenvironment of a cell, but the outer membrane is susceptible to agentsthat compromise membrane integrity. Pseudomonas aeruginosa cells treatedwith chloroform (which compromises the outer membrane), then withpurified GP36 CD fragment. The GP36 CD fragment resulted in a rapid,concentration-dependent reduction in OD (lysis of the bacteria). Furthertests and controls were performed to confirm activity, using time courseof killing and titration of amount of GP36 CD. Bacterial lysis dependedupon the chloroform treatment, and confirmed that the muralytic domaincan kill the Gram-negative target once it reaches the peptidoglycanlayer.

C. Bacterial Targets

As indicated above, the peptidoglycan layer in most Gram-negativebacteria is relatively thin. The GP36 CD construct was tested on thepeptidoglycan of different clinically relevant species of Gram-negativetarget bacteria. Similar experiments were performed on Acintobacterbaumanii, Klebsiella pneumoniae, E. coli, Salmonella typhimurium,Salmonella gallinarum, Salmonella enteritidis, and Salmonella dublin.The GP36 CD similarly resulted in a rapid, concentration-dependent lysisof chloroform treated bacteria of all tested species.

Example 2. Membrane Transfer Domains A. Chimera Comprising MuralyticDomain and Membrane Transfer Domain

To access the peptidoglycan layer in a normal Gram-negative bacterialtarget, a physiologically compatible means for penetrating the bacterialouter membrane barrier was considered. In particular, the penetratingactivity must be specific for the bacterial outer membrane while nothaving significant negative effect on eukaryotic cells.

The GP36 CD enzymatic segment was linked to the membrane transfer domainderived from the human bacterial permeability increasing (BPI) protein.The human BPI nucleic acid sequence is NM_001725.2 (see SEQ ID NO: 3),which encodes the protein sequence BAG37729 (see SEQ ID NO: 4). Theconstruct was designed with a 22 amino acid N-terminus extensionincluding the poly-His (for purification), followed by residues 683-898of GP36, linked via three arginine residues to amino acids 16-39 of theBPI TMD segment (SEQ ID NO: 4), followed by an additional 3 C-terminalarginine residues. TMD is the designation for the segment with membranepermeating activity, e.g., the transmembrane domain. The polynucleotideconstruct is designated SEQ ID NO: 8, which encodes the polypeptidedesignated P225 (SEQ ID NO: 9).

As described above, the expression construct was introduced into E.coli, production of protein was induced, and the cells harvested. Thecell pellets were processed to isolate the protein, which was mostlylocated in inclusion bodies. The protein was solubilized, and theproduct was purified using Ni-NTA column chromatography. The protein wasgreater than 95% pure by SDS-PAGE.

B. Bacterial Killing Assay

Pseudomonas auruginosa cells were exposed to the purified P225, whichresulted in rapid, concentration dependent OD reduction (i.e., bacteriallysis) in the absence of additional permeating agents (e.g.,chloroform). Further tests and controls were performed to confirmactivity, titrating amount of protein at a time point of one hour.Bacterial killing was observed using a different CFU reduction assay.The killing of live Gram-negative bacteria demonstrates that the BPI TMDcan successfully provide the GP36 enzymatic domain access to thepeptidoglycan layer inside the bacterial outer membrane.

C. Other Bacterial Targets

The P225 construct was tested for activity on the peptidoglycan ofdifferent clinically relevant species of Gram-negative target bacteria.Similar experiments were performed on additional strains of Pseudomonasauruginosa, E. coli, Klebsiella (which is highly resistant to manyantibiotics), and Acinetobacter baumanii, all of which were effectivelykilled.

Example 3: P266 Construct and Biological Activity

SEQ ID NO: 10 represents a variant of P225, designated P266 (encoded bySEQ ID NO: 11). The N terminal Met can be removed so that thepolypeptide begins with Gly. Relative to P225, P226 has a shorter, 6N-proximal His tag, and lacks the segment following histidines. Thisprovided a construct having segments: 6×His tag-GP36 CD-RRR-BPI TMD-RRR.The GP36 CD would run from about Gly(9) to Glu(224), the first RRRcorresponds to R(225) to R(227), the BPI TMD corresponds to Ala(228) toR(251), and the final RRR corresponds to residues 252-254. The projectedmolecular weight is about 27.6 kDa, with a pI of about 9.48. Thisincludes the N terminal Met, which can be removed.

Like the P225 construct, P266 was insoluble upon expression in E. coliBL21 (DE3) cells after induction with IPTG. The induced cell pellet wasresuspended in lysis buffer (50 mM Tris base, 0.1M NaCl, 0.1%TritonX100), and sonicated using a 13 mm probe for 10 minutes. Thesonicated cell pellet was centrifuged at 16,000 rpm for 10 minutes andthe inclusion bodies pellet collected. The inclusion body pellet wassolubilized by resuspending the pellet in Buffer A (6M GuHCl, 100 mMNaH₂PO₄. 10 mM TrisCl, pH 8.0) and kept rocking for 30 min at roomtemperature. The ratio of IB: buffer volume was 1 gram wet weight of IBwith 40 ml of bufferA. The solubilized proteins were centrifuged at16,000 rpm for 10 min and the clear supernatant was collected. Ni-NTAmatrix was equilibrated with Buffer B (8M urea, 100 mM NaH₂PO₄, 10 mMTrisCl, pH 8.0) with 5 column volumes used for equilibration. Thesolubilized clear supernatant was loaded on to the equilibrated Ni-NTAcolumn and allowed to pass through in gravity mode and the flow throughcollected. The column was washed with 10 column volumes of Buffer B toremove impurities and unbound proteins. It was then washed with 10-15column volumes of Buffer C (8M urea, 100 mM NaH₂PO₄, 10 mM TrisCl, pH6.5). The protein elutions were carried out in Buffer E (8M urea, 100 mMNaH₂PO₄, 10 mM TrisCl, pH 4.5). Fractions were collected and analyzed bySDS PAGE. Fractions containing protein of interest in high amounts asseen on SDS PAGE gels were pooled and dialyzed in a stepwise manner.

Dialysis was carried out against a buffer volume˜100 times of the pooledeluate volume (e.g., 10 ml eluate dialized against 1 liter buffer), inthree steps, first against 4M Urea in 20 mM sodium phosphate buffer, pH6.0, for 5 hrs at 4deg C.; second against 2M urea in 20 mM sodiumphosphate buffer, pH 6.0, for 5 hrs at 4deg C.; and third against 20 mMsodium phosphate buffer, pH 6.0, with 5% sucrose, 5% sorbitol, and 0.2%Tween 80, for 5 hrs at 4 deg C. The sucrose, sorbitol, and Tween80components help stabilize the protein from aggregation andprecipitation. Eluates taken out post dialysis were centrifuged toseparate any precipitation. The cleared supernatant was collected andprotein content estimated for activity assay. The final product wasabout 85-95% homogeneous by SDS PAGE with coomassie blue staining andsilver staining.

The purified protein was assayed for bacterial killing using a CFU dropassay and typically simultaneously monitored for residual OD600 at theend of 16 hours of treatment with the protein product. Log phase PA01Pseudomonas aeruginosa target cells were resuspended in a suitablebuffer at an absorbance of 1.0, which corresponds to about 1E7 cells.The protein was tested at 50 μg in either acetate or glycine buffers.The assays were performed in 20 mM sodium phosphate buffer (pH 6.0), 5%sucrose, 5% sorbitol, and 0.2% Tween80 with either 20 mM sodium acetate(pH 6.0) or 50 mM glycine-NaOH (pH 7.0) at 37° C. for 2 hrs at 200 rpmagitation.

The CFU drop assay in sodium acetate buffer provided about 5 log drop,and in the glycine buffer provided at least 7 logs drop after treatmentwith the protein. From the residual OD600, the acetate buffer providedabout 80% less in comparison to control, while the glycine bufferprovided about 95% residual decrease in comparison to control.

The CFU drop assay in glycine buffer (pH 7.0) was evaluated without thesucrose, sorbitol, and tween80 stabilizers in the incubation. The CFUdrop without stabilizers was the same with stabilizers in the assay, atleast 7 logs drop. In many cases, other stabilizers or additives may beuseful or important. These may include materials such as polyols, e.g.,sorbitol and related compounds; glycerols, e.g., in the range of 0-10%;sugars, such as sucrose, e.g., in the range of 0-5%; detergents orsurfactants such as Triton X100, Brij 35, NP-40, Tween 20,Octylbetaglucoside, Sarkosyl, Tween80, etc., preferably tween80, e.g.,in the range of 0.1% to 0.5%; and metal chelators such as EGTA, EDTA,preferably EDTA, e.g., in the range if 50 μM-100 μM.

The biological activity of the P266 was titrated across proteinconcentration on the PA01 target strain. Both the CFU drop and theresidual OD600 progressed with 2 hr incubations as the protein wasincreased from 5, 10, 25, and 50 μg protein. Under the conditionstested, both by CFU drop and residual OD600, with 50 μg P266 at 37° C.and 2 hr incubation, treatment could kill virtually all cells at 1E6 and1E7 cells in the assay, but showed much decreased killing with 1E8 ormore cells in the assay. Incubation time over the 1-4 hour range did notseem to have dramatic effects on PA01 killing assays.

Testing stability of the P266 at various temperatures, the proteinappeared to maintain killing activity after 1 hr exposure to 37°, 42°,and 65° C. The product appears to be relatively heat stable up to 65° C.for an hour.

Testing target killing efficiency, the P266 had substantial killingactivity, by both the CFU drop and OD600 drop assays, on Pseudomonasaeruginosa, NDM1 plasmid carrying Klebsiella pneumoniae, NDM1 plasmidcarrying E. coli, Klebsiella pneumoniae, Acinetobacter baumanii,Salmonella typhimurium, Salmonella infantis, and E. coli isolates. P266had less killing activity on Shigella, Proteus mirabilis, andBurkholderia thailandensis isolates using the same conditions andconcentrations. Similarly, P266 killing activity on Gram-positivebacterial strains was lower than with P. aeruginosa, but could likely beincreased with longer incubation or higher concentration. The resultsshow, however, that P266 has a broad range of target bacteria, i.e.,broader than the target range for any known phage.

The effect of P266 incubation with human red blood cells was minimal atthe highest tested 25 and 50 μg amounts. With 1 hr incubations, redblood cells maintained integrity, e.g., containing hemoglobin, and thecells could be sedimented into pellets. P266 therefore does not disrupteukaryotic cell membranes, suggesting compatibility with in vivo uses ofthis protein product.

Example 4: Soluble P266 Variant: P275

P266 is insoluble when expressed in E. coli, making productiondifficult. This insolubility requires protein purification anddenaturation to solubilize, and refolding can lead to significant lossesof active protein. In addition, protein oxidation can further reduceactivity.

A variant of P266 was designed to decrease the hydrophobicity in the BPIsegment (the 23 amino acid MTD). The aim was to subtly disrupt thefolded structure of the protein to expose more of the hydrophobicinterior to aqueous solution, e.g., removing the shells of watermolecules that form over the hydrophobic patches on the surface of somefolded proteins. In particular, a nucleic acid construct was designed togenerate a variant protein from the P266, designated P275, withconversions of V232 to E; V234 to D; and I236 to K. See SEQ ID NO: 12and 13.

FIG. 1 shows a comparison of DAS plots of P266 and P275. FIG. 2 shows acomparison of the TMHMM plots of P266 of P275. The relative GRAVY scoresfor the BPI domain and variants thereof are shown below.

BPI TMD GRAVY SCORE Wild Type Sequence: ASLMVLVAIGTAVTAAVNPGVVVR: 1.658Variants ASLMELDAKGTAVTAAVNPGVVVR: 0.667 ASLMKLKARGTAKTAAKNPGKKRR:−1.104 ASLMKLKARGTAKTAAKNPGKVRR: −0.767 ASRMVLVARGTAKTAAVNPGVVRR: 0.27

P275 exhibits a number of surprising and unexpected properties. Theexpression construct was expressed in E. coli BL21(DE3) with inductionat 37° C., 1 mM IPTG, as was the P266 expression. P275 did not, however,form inclusion bodies, and the majority of the protein product wasrestricted to the soluble fraction. Moreover, and unexpectedly, thesoluble protein did not traverse the bacterial cell membrane to accessthe peptidoglycan layer (located in the periplasmic space) and kill theGram-negative E. coli production cell host. The results show that MTDscan be manipulated to be soluble when expressed in the cell but notcapable of traversing the bacterial cell membrane. The manipulated MTD,however, retains the function of allowing the construct to traverse theouter membrane of a Gram negative bacteria. Thus, the manipulatedMTD-muralytic domain chimera can access the sensitive peptidoglyan layerof Gram-negative bacteria.

The soluble P275 product was much simpler to handle in purification andrecovery, and provided much higher yields of active protein. The solubleP275 protein was purified on the Ni-NTA column at pH 8.0; eluted withimidazole at pH 4.5, dialyzed to remove imidazole, and reformulated intoassay buffer. The P275 induced cell pellet was resuspended in Lysisbuffer (50 mM Tris Base, 0.1M NaCl, 0.1% TritonX 100) and sonicated. Thesonicated cell pellet was centrifuged 16,000 rpm for 10 min, and thesupernatant collected and pH adjusted to 8.0. A Ni-NTA matrix wasequilibrated with (50 mM Tris.Cl, pH 8.0) using 5 column volumes. Thesolubilized protein was loaded on to the equilibrated Ni-NTA column andallowed to pass through. The flow through was collected and passedthrough the column once again. The column was washed with 10-15 columnvolumes of 20 mM sodium phosphate buffer, pH 6.5, then washed with 5column volumes of 20 mM sodium phosphate buffer, pH 4.5. Protein elutionwas carried with IM imidazole in 20 mM sodium phosphate buffer, pH 4.5.Eluted fractions were collected and analyzed by SDS PAGE. Fractionscontaining the protein of interest in high amounts as seen on SDS PAGEgels were pooled and dialyzed. Dialysis was carried out against a buffervolume ˜100 times of the pooled eluate volume, three changes against 20mM sodium phosphate buffer, pH 6.0 each for 5 hrs at 4 deg C. Eluatestaken out post dialysis were centrifuged to separate any precipitation,and the supernatant collected and additives sucrose, sorbitol, andTween80 were added to a final concentration of 5%, 5%, and 0.2%respectively. Protein content was estimated for activity assays.

The P275 product is soluble and easy to purify, which allows a more costeffective downstream operation avoiding the requirement for denaturingagents, and achieving about 85% purity in a simple process leading to abiologically active product. Moreover, and surprisingly, P275 had acomparable or better activity in a CFU drop assay under standard 50 ugprotein amounts at 37° C. with 2 hr incubation times.

Example 5: Effect of Oxidation on Chimeric Proteins (e.g., P275)

P275 was tested to see whether protein oxidation might be affectingbiological activity. Two amino acids most prone to oxidation are Cys andMet. P275 has no Cys residues, but the Met may be subject to oxidation.Assay in the presence of methionine or sodium thiosulfate could minimizeprotein oxidation. When the CFU drop assays were performed on the P275protein in the presence of 0.05% methionine, or 0.1% sodium thiosulfate,the CFU drop was substantially greater than in the absence of eitheragent. Thus, a formulation of a chimeric protein as described hereinwith an agent which reduces oxidation, e.g., methionine, sodiumthiosulfate, or ascorbic acid, from the range of 0.001-0.2%, 0.001-0.3%and 1-100 mM respectively, e.g., 0.05%, 0.1% and 5 mM of these agents,can be added to retain biological activity, e.g., in storage or assayconditions where oxidation can occur.

To minimize the oxidation which may occur in the protein, the metresidues may be removed/replaced, e.g., by site specific mutagenesis.This is relatively simple and straightforward, as described for alaninescanning mutagenesis. See, e.g., Kristensen, et al. (1997) J. Biol.Chem. 272:12978-12983. Substituting one or more Met with another lesssusceptible amino acids e.g., threonine, minimizes the oxidation effectson the protein construct. The construct can be tested initially forsusceptibility to loss of activity due to oxidation before embarking onsite-specific mutagenisis.

The following references provide additional guidance for addressingstability issues related to oxidation: Yokota, et al. (2000) J.Pharmaceutical and Biomedical Analysis 24:317-324; Grune, et al. (1997);FASEB J.11:526-534; Lam, et al. (2003) J. Pharmaceutical Sci.86:1250-1255; Kenley and Warne (1994) Pharmaceutical Research 11:72-76.

Example 6: Substitution of BPI with Alternative MTDs

Table B lists MTD segments which can be combined with catalyticmuralytic segments listed in Table A. In P225, P266, and P275, thecatalytic domain is N-proximal to the MTD. The relative positions of thetwo domains can be reversed with the MTD N-proximal to the catalyticdomain. If included, the positively charged segment, e.g., RRR, KKK,NNN, QQQ, or mixtures of these positively charged amino acids, aretypically adjacent to the MTD, e.g., to attract the MTD to thenegatively charged outer membrane.

Sequences of the appropriate domains are available from GenBank and theliterature, and appropriate constructs can be created to producechimeric expression vectors. Chimeric expression constructs can begenerated with various combinations of the domains (e.g., a selectedsubset of the domains in Tables A and B). Once the combinations areconstructed, the expression vectors can be introduced into appropriateexpression hosts. Upon expression, the products can be tested with theCFU drop or OD600 drop assays for biological activity on targetbacteria. Products with activity can be modified or optimized foractivity parameters, protein stability, target specificity, criticalresidues or boundaries of domains, and other features as shown.

For example, as a substitute for BPI, the membrane translocating domain(MTD) from a Pseudomonas phage P134 holin can be used for MTD functionwith a selected catalytic domain, e.g., the GP36 segment. Described areconstructs for an additional 5 alternative MTD segments besides the BPIdescribed above.

Five substituted MTD constructs as described below:

-   A. 10×His-GP36 CD-RRR-P134 holin MTD-RRR-   B. 10×His-GP36 CD-RRR-LPS BP peptide-RRR-   C. OP36 CD-T4 phage gp5 beta helix-LE-6×His-   D. GP36 CD-S type pyocin OM binding domain-LE-6×His-   E. GP36 CD-P22 tail spike-LE-6×His

A. P134 Holin TMD: (SEQ ID NO: 14 and 15)

Phage P134 holin transmembrane domain was derived from the holin gene ofphage P134. The MTD from P134 holin was identified based on transmembrane helix prediction programs, DAS, and TMHMM. The 23 as TMD iscloned C terminal to GP36CD with linker and terminal arginines. TheGP36CD-RRR-P134TMD-RRR is cloned as an N terminal 10×his tag. Anconstruct of 804 nucleotides, GP36CD-RRR-P134 holinTMD-RRR with 10×Histag and additional vector sequence derived from vector, is described inSEQ ID NO: 14.

-   NT 1-66 encode the vector-10×His; residues 1-22 (Met is removed in    prokaryotes)-   NT 67-714 encode the GP36 muralytic domain; residues 23-238-   NT 715-723 encode RRR linker; residues 239-241-   NT 724-792 encode the P134 holin MTD; residues 242-264-   NT 793-801 encode the C terminal RRR; residues 265-267-   NT 802-804 encode termination codon

Highly hydrophobic residues in the P134 MTD to target for modificationare shown as underlined residues substituting wild type amino acids inthe “variants” below. Wild type residues that can be substituted toincrease solubility include Ile243; Leu246, Ala248, Ala249, Val250Val256, Ile261, and Leu264 (see SEQ ID NO:15). The residues are numberedaccording to SEQ ID NO: 15, which includes the N-terminal Met which isremoved in production. Replacement amino acids will typically be aminoacids with side chains having similar size. Examples of substitutionsinclude but are not limited to: ile to arg, asp, asn, or lys; leu topro, arg, or lys; val to asp, lys, or arg, and ala to lys.

P134 TMD GRAVY SCORE Wild Type Sequence: EIASLCAAVLTALYVGAQLITLL: 1.774Variants: EIASLCAARPTALYVGAQLITLL: 1.161 ERASLCAARLTALYRGAQLRTLL: 0.235EKASLCKKRRTALYKGAQLDTLL: −0.526 EIASRCAAVLTALYVGAQLNTLK: 0.730

B. Lipopolysachharide Binding Protein Peptide (SEQ ID NO: 16 and 17):

A stretch of 30 amino acids involved in binding to LPS of gram negativebacteria was identified and selected as an alternative MTD. See Horwitz,Williams, and Nowakowski (1995) Infection and Immunity 63:522-527.Derived from this report; Protein ID: CAA67226 (AA 106 to 135). Thissequence is fused to GP36 catalytic domain (CD) to generate the 825nucleotide construct which encodes molecule GP36CD-RRR-LBP peptide-RRR.This is cloned with an N terminal histidine tag. The construct is 825nucleotides, predicted 274 residues (including N terminal Met, whichshould be removed in prokaryote hosts) Mw: 30.38 kDa, and pI: 9.72.

-   NT 1-3 is initiation MET, removed in most prokaryote hosts-   NT 4-66 encodes the vector segment-10×His; residues 2-22-   NT 67-714 encodes the GP36 muralytic domain; residues 23-238-   NT 715-723 encodes the RRR linker, residues 239-241-   NT 724-813 encodes the LPS BP MTD; residues 242-271-   NT 814-822 encodes the RRR linker; residues 272-274-   NT 823-825 encodes termination codon

The LPS Binding Protein derived MTD is described in SEQ ID NO: 17. Theresidues which are indicated for replacement to generate a more solublevariant include Val248; Val267; Val269; Phe258; and Phe259.

C. T4 Phage gp5 Beta Helix (SEQ ID NO: 18 and 19):

The C terminal beta helix domain from protein gp5 of phage T4 isreported to be responsible for penetrating the outer membrane of abacterial cell during infection. See Leiman, et al. (2010) Virology J.7:355. The gp5 beta helix sequence (187 aa; from NP_049757.1 residues389-575) is cloned C terminal to GP36CD with a C terminal 6× histidinetag. The construct is 1245 nucleotides; predicted 414 residues withpredicted pI/Mw: 5.43/45353.99 with N terminal Met uncleaved.

-   NT 1-651 encodes the initiation MET and GP36 muralytic domain;    residues 1-217-   NT 652-657 encodes a KL (generated by restriction enzyme site Hind    III); residues 218-219-   NT 658-1218 encodes the T4 phage gp5 beta-helix; residues 220-406-   NT 1219-1224 encodes a LE (generated by restriction enzyme site    XhoI); residues 407-408-   NT 1225-1242 encodes 6×His; residues 409-414-   NT 1243-1245 encodes termination codon

D. S Type Pyocin Outer Membrane Translocation (OMT) Domain (SEQ ID NO:20 and 21):

The outer membrane translocation domain from S type pyocins translocateacross the outer membrane of P. aeruginosa. See Ling, Saeidi, Rasouliha,and Chang (2010) “A predicted S-type pyocin shows a bactericidalactivity against clinical Pseudomonas aeruginosa isolates throughmembrane damage” FEBS Letters 584:3354-3358. The OMT domain (181residues; see Protein Acc. No. EHS39709, residues 217-397; P. aeruginosaMPAO/P1) was cloned C terminal to GP36CD with histidine tag at the Cterminus. The construct of 1227 nucleotides encodes 408 amino acids(including the N terminal Met, which is typically removed by prokaryotehosts), which has a predicted pI/Mw: 8.44/44543.37

-   NT 1-651 encodes the initiation MET and GP36 muralytic domain;    residues 1-217-   NT 652-657 encodes a KL (generated by Hind I site); residues 218-219-   NT 658-1200 encodes the OM translocation domain; residues 220-400-   NT 1201-1206 encodes a LE (generated by restriction enzyme site    XhoI); residues 401-402-   NT 1207-1224 encodes 6×His; residues 403-408-   NT 1225-1227 encodes termination codon

E. P22 Tail Spike Protein (SEQ ID NO: 22 and 23):

The tail spike protein of phage P22 has endorhamnosidase activitycapable of modifying O-antigen, hence, compromising the structure oflipopolysaccharide of Salmonella. See, e.g., Steinbacher, et al. (1997)“Phage P22 tailspike protein: crystal structure of the head-bindingdomain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56A resolution, and the molecular basis of O-antigen recognition andcleavage” J Mol Biol. 267:865-80; and Waseh, et al. (2010) “OrallyAdministered P22 Phage Tailspike Protein Reduces Salmonella Colonizationin Chickens: Prospects of a Novel Therapy against Bacterial Infections”PLoS One 5(11): e13904. The Salmonella tail spike protein (see ProteinID: NP_059644, amino acids 2-667) was cloned C terminal to GP36CD withhistidine tag at the C terminus. The theoretical size is 893 aminoacids; pI/Mw: 5.76/96694.40.

-   NT 1-651 the initiation MET and GP36 muralytic domain; residues    1-217-   NT 652-657 encodes a KL (generated by restriction enzyme site Hind    III), residues 218-219-   NT 658-2655 encodes the P22 tail spike protein domain, residues    220-885-   NT 2659-2664 encodes a LE (generated by restriction enzyme site    XhoI), residues 886-887-   NT 2665-2682 encodes 6×His; residues 888-893-   NT 2683-2685 encodes termination codon

Example 7 Expression, Purification, and Validation of Chimeric Proteins

The following descriptions are examples of how to prepare and validatethe additional chimeric constructs. Buffers, media, times, and otherconditions can be modified as will be understood by one of skill in theart of protein expression and design.

Codon optimization for expression in E. coli (codon bias), changing theGC content, incorporating alternate fusion tags (e.g., glutathioneS-transferase GST), nusA transcription elongation factor, maltosebinding protein (MBP), intein, among many possibilities), varyinginducer concentrations, temperature, expression with chaperones forimproved folding, and different expression hosts can be used to improveexpression of the desired constructs.

For example, competent cells of a production host, e.g., E. coli, aretransformed with the respective plasmid, plated on LB+ampicillin (100μg/ml) or kanamycin (20 μg/ml), and incubated overnight at 37 deg C. Thecultures from plates are scraped into LB+antibiotic, typically liquid,and grown to OD₆₀₀˜0.8 to 1.0. The cells are then induced with IPTO at 1mM and incubated at 37 deg C. for 4 hours. The cells are harvested bycentrifugation at 8000 rpm for 10 minutes and the pellet stored at −80deg C.

Where constructs accumulate in inclusion bodies, the induced cell pelletis resuspended in lysis buffer (50 mM Tris base, 0.1 M NaCl, 0.1%TritonX100), and sonicated using a 13 mm probe for 10 minutes. Thesonicated cell pellet is centrifuged at 16,000 rpm for 10 minutes and apellet containing inclusion bodies (IB) is collected. The inclusion bodypellet is solubilized by resuspending the pellet in Buffer A (6M GuHCl,100 mM NaH₂PO₄. 10 mM TrisCl, pH 8.0) and kept rocking for 30 mins atroom temperature. The ratio of IB: buffer volume is typically I gram wetweight of IB with 40 ml of buffer A. The lysate is centrifuged at 16,000rpm for 10 min and the clear supernatant is collected. A Ni-NTA matrixis equilibrated with Buffer B (8M urea, 100 mM NaH₂PO₄, 10 mM TrisCl, pH8.0) with 5 column volumes used for equilibration. The supernatant fromthe IB is loaded on to the equilibrated Ni-NTA column and allowed topass through in gravity mode and the flow through is collected. Thecolumn is washed with 10 column volumes of Buffer B to remove impuritiesand unbound proteins. The column is then washed with 10-15 columnvolumes of Buffer C (8M urea, 100 mM NaH₂PO₄, 10 mM TrisCl, pH 6.5). Theattached protein elutions are carried out in Buffer E (8M urea, 100 mMNaH₂PO₄. 10 mM TrisCl, pH 4.5). Fractions are collected and analyzed bySDS PAGE. Fractions containing protein of interest in high amounts asseen on SDS PAGE gels are pooled and dialyzed in a stepwise manner. Thepooled fractions are subject to dialysis carried out against a buffervolume ˜100 times of the pooled eluate volume (e.g., 10 ml eluatedialized against 1 liter buffer). The dialysis is performed firstagainst 4M urea in 20 mM sodium phosphate buffer, pH 6.0, for 5 hrs at4deg C.; then secondly against 2M urea in 20 mM sodium phosphate buffer,pH 6.0, 5 hrs at 4deg C.; and thirdly against 20 mM sodium phosphatebuffer, pH 6.0 with 5% sucrose, 5% sorbitol, and 0.2% tween80 for 5 hrsat 4 deg C. Eluates taken out post dialysis are centrifuged to separateany precipitated material. The cleared supernatant is collected andprotein content estimated for activity assay.

To verify activity (bacterial cell killing activity) a CFU drop assaycan be performed as follows. Bacterial cells are grown in LB broth toabsorbance at 600 nm reaches a range of 0.8 to 1.0. Then 1 ml of cultureis spun at 13000 rpm for 1 minute and supernatant discarded. The cellpellet is resuspended in one ml of 50 mM Glycine-NaOH buffer (pH 7.0)and cell numbers adjusted to about 1×10⁸/ml. Test protein is added to100 μl cells to achieve final concentration of about 50 μg and volumemade-up to 200 μl with 20 mM sodium phosphate buffer (pH 6.0) withadditives. The protein is incubated with cells at 37 deg C. for 2 hourswith 200 rpm agitation, then the samples are log diluted in LB broth andplated on LB agar to quantitate residual CFU. The plates are incubatedat 37 deg C. overnight for colonies to grow.

The Metabolic Dye Reduction assay can also be used to determine livecell numbers. The assay is based on the principle that viable cellsreduce Iodo-Nitro Tetrazolium (INT), a metabolic indicator dye. Briefly,1×10⁷ target cells, e.g., P. auruginosa, in 100 μl volume are mixed withtest protein in 100 μl to achieve final concentration of about 50 μg andvolume made-up to 200 μl with 20 mM sodium phosphate buffer (pH 6.0)with additives in microtiter plate wells. A cell control is alsomaintained. Samples are incubated at 37 deg C. with 200 rpm for 2 hourand INT dye (1×) is added to all samples. The microplate is incubated indark at room temperature for 20 minutes and the absorbance at 492 nm isrecorded. 10×INT stock solutions are prepared by dissolving 30 mgTetrazolium Violet (Loba Chemie, India) in 10 ml of 50 mM SodiumPhosphate buffer, pH 7.5.

Example 8: Detection of Bacterial Cell Binding

In Gram-negative bacteria, the outer membrane (OM) and the peptidoglycanare linked by lipoproteins. The OM includes porins, which allow thepassage of small hydrophilic molecules. See, e.g., Cabeen andJacobs-Wagner (2005) “Bacterial Cell Shape” Nature Revs Microbiology3:601-610; Nikaido (2003) Microbiol. Mol. Biol. Rev. 67:593-656. Thestructure and composition of the outermost layer of the cells isreported to be different between different bacteria. On the outerenvelope cells may have polysaccharide capsules (see, e.g., Sutherland(1999) Biotechnol. Genet. Eng. Rev. 16:217-29; and Snyder, et al. (2006)Carbohydr. Res. 341:2388-97.) or protein S-layers (Antikainen, et al.(2002) Mol. Microbiol. 46:381-94; Schäffer and Messner (2005)Microbiology. 151:643-51; and Avall-Jääskeläinen and Palva (2005) FEMSMicrobiol Rev. 29:511-29), which protect bacteria in unfavourableconditions and affect their adhesion. The basic structure oflipopolysaccharide (LPS), a covalently linked lipid andheteropolysaccharide, is common to all LPS molecules studied, but thereare variations depending on bacterial genera, species, and strains. See,e.g., Treat, et al. (2006) J. Endotoxin Res. 12:205-23; Raetz andWhitfield (2002) Ann. Rev. Biochem. 71:635-700; Yethon and Whitfield(2001) Curr. Drug Targets Infect. Disord. 1:91-106; and Yethon andWhitfield (2001) J. Biol. Chem. 276:5498-504.

The chimeric constructs described herein can thus be tested for bindingto a target bacteria. Described here are various assays for whether theconstruct (with MTD) reaches the peptidoglycan layer. SDS-PAGE can beused to detect binding of the protein to cells. For example, 10⁷ cellsare treated with a suitable amount of protein for approximately 2 hours.Then the cells are pelleted by centrifugation and the amount of proteinin the supernatant is examined on SDS-PAGE and stained. The protein islabeled as adsorbed to cells, if the intensity of the protein before theadsorption to cells is higher than the one after adsorption, thedifference is likely to be due to cell binding. Binding can also bedetected using, e.g., confocal imagin to detect changes to the bacterialOM upon exposure to a chimeric construct. Fluorescent tags or luciferasecan also be used, as will be recognized by one of skill.

INFORMAL SEQUENCE LISTINGSEQ ID NO: 1 (P134 GP36 full length DNA sequence: highly homologous to GeneID 1482616)SEQ ID NO: 2 (AA translation of SEQ 1 (GP36); highly homologous to NP877475)SEQ ID NO: 3 (Homo sapiens cDNA, FL096367, highly similar to Homo sapiensbactericidal/permeability-increasing protein (BPI), mRNA; GenBank:AK315328.1) SEQ ID NO: 4 (BPI; unnamed protein product [Homo sapiens]GenBank: BAG37729,1)SEQ ID NO: 5 (GP36 CD nucleic acid; first CHC13 test construct)SEQ ID NO: 6 (24 Kda construct; translation product of SEQ ID NO: 5, 217amino acids)SEQ ID NO: 7 [13 aa C terminal extension attached for purification purposesonto 24 Kda]SEQ ID NO: 8 [encoding chimeric GP36 segment linked to BPI segment; P225]SEQ ID NO: 9 [linker-GP36 muralytic domain-RRR-BPI TMD-RRR; P225polypeptide]SEQ ID NO: 1 (P134 GP36 full length DNA coding sequence: [close homolog to Gene ID1482616]) Nucleotide sequence of P134 GP36: 1ATGGCGGAATCGCAACGTGCTTCCCAAGAGCTTGGGATCAACGTCGGACAGGCGCAACTC 60 61CAGCCGGGCCAGAGTGCCCGGCGCGGAGTGCGCGACTCCGAGGTCAACTACAGCGGTCCG 120 121AGTGTAGGCTCGCAGATTCTCGACGGCATCCTGGGTGCCGGTCAGCAGATCGCTGGCAAA 180 181TGGTTCGAGCACAACGTGCAGCAGGAAGTTCTGCGCGGTGAGCGTGCCCGTATGGCCGGC 240 241GAGGCTGAGGAGGCAGTAGACAGCAACGTACTGGCCAAACCATTCGTGAAGGGTGGTTGG 300 301CGTAAGCAGGACTACCGTATCGCCCAGGCGGACTTCAGCCTGAAGATGCAGCGATTCATC 360 361GCCAACAAGGGCCGGGAGATGACTCCCGAGGAGTTCCGCAAGTACCTGTCCCAGGAGGCT 420 421ACGCACGTCCTGGACTCGACCGAGGGCATGAACCCCAACGATGCCCTACAGGCGATGGCA 480 481CAGCAGCATAAGGCCGAGGAACAGCTCTTTGGCATGCAGGCTAAGGCGTACATGGATTGG 540 541TCCATCGACCAGGCCGCACGGGGCTTCCGCACCCAGGGTAACAGCATCCTGGCCAAGGCC 600 601GTACATGCCCAGGCCACCGGCGACGAGCTATCCCGGCAACTCAGCCTGGAAGAGGCCGGC 660 661CTGTTCTATACCAACATCATGACCTCCGAGGATATCCCGCTGGAGGTACGTGACAAGGTG 720 721GGTATGCAGTTCCTGGCGGCCAGCCTGGACATGAACCAGCGGGGCATCTATGAGGGCCTG 780 781CGCGATGCCGGCTTCCTGGACAGTATGTCCTTTGACGACCGGCGTGCGCTCAACGGCCTC 840 841TATGAAAAATCGAAGGCACAGACCCGTGCCAAGGAATCGATGGCTACCCTGCGGGCCGAC 900 901GCCGACTTCCAGCAGCGGGTGGCCAACGGCGCCATCACAGACCTTGCCGAGGTTGAGGCG 960 961TACTCACGAGGCATGGTCGAGGAGGGCCGCTGGAGCGACGCTCAGGCCATCTCGTTCATG 1020 1021ACCAAGGCCATGACCGGCCTGGGCAACGCTCAGCGCATGCAGGGCATCATGGCGGCCTTG 1080 1081GAAGCCGGAGACATCAACGCCCTCCACACGCTGGGTACCAACGTCACCGAGGCGCTGGAG 1140 1141CAGTGGGACAAGATGCAGGCCGCCAACGGCTCAAGCCTGACTGACCGTCTCGTGCAGGGC 1200 1201ACACAGCTCGGCCTGCGCCTGGGGACCTTCCCCAAGACCTACGGCGAGTCCGTGGGCAGC 1260 1261GCGGTGCGCATGATCCAGGCCGCCAAGGAAGGCGAGGCAAACCCGGAGCTGGTCAACACG 1320 1321CTGAACAGCATCTTCGAACAGGTGGCCTCGGCCCAGGAGATCAACCCATCCGCCGGCAAC 1380 1381GTGATGCTATCCGGCATCCCGGAAGCCGAGCAGGGCGCCGTGGCCTGGGCACTCAAGCAG 1440 1441ATGAAGATGGGCATCGCACCAGCTCAACCTCTGCGCGAGTTTAGCGCCAACGCCGAAGTC 1500 1501GTGAAGCAGATGGACGAGTTCGAGAAAGGCCAGAACACCAAGGCATTCAAGGACAACCTC 1560 1561GGTAAGCAGGTCAACGACAAGTTCGTGAACAACATCTTCGGTCGAGCCTGGAACATGCTG 1620 1621ACCGGCGAGAGCGACCTGAGCAACAACGAGGCCGTCCTGAGCATGTATCGCCGGGCGACC 1680 1681ATCGACGAGGCGAACTGGCTGGCCAGCGACCGCAAGCATGCGGGTCTGCTCACCAGCGAC 1740 1741ACGGGCCGCGAGGCCCTGCTGGAGATCGCCGCCGCCAACGTGCGTAACCGCACCATCCAG 1800 1801GTAGGCGAAGGTCGGAACCTGAAGGAAGGGGACCTATTCAGCCGCCGCGATAGCGCGCCG 1860 1861CTGATCCTGCCTCGCGGCACCACCGCCGAGCAGCTATTCGGGACCAACGACACCGAGACC 1920 1921ATCGGAACCGTCCTGGCCGAGCAGCACAAGCCGCATGTCGAAGGACTCCTCGGCTACAAG 1980 1981TCGGTAGTCGCCTTCGAGTACGACCGCACCAGTGGCAGCCTCCTCGCCGTCGAGTACGAC 2040 2041GAGAACGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTG 2100 2101CTCAAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTC 2160 2161AAGGTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAG 2220 2221GACGTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTAT 2280 2281AAGGATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGG 2340 2341GCAGGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGC 2400 2401GCACTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATC 2460 2461CTGGGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAAC 2520 2521ACCTTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGC 2580 2581AAGTGGTACACGCAGACGCCCACCGGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCAC 2640 2641TTCGATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGTAA 2697SEQ ID NO: 2(AA translation of SEQ 1 (P134 GP36); highly homologous toNP877475) Amino acid sequence of P134 GP36: 1MAESQRASQELGINVGQAQLQPGQSARRGVRDSEVNYSGPSVGSQILDGILGAGQQIAGK 60 61WFEHNVQQEVLRGERARMAGEAEEAVDSNVLAKPFVKGGWRKQDYRIAQADFSLKMQRFI 120 121ANKGREMTPEEFRKYLSQEATHVLDSTEGMNPNDALQAMAQQQKAEEQLFGMQAKAYMDW 160 181SIDQAARGFRTQGNSILAKAVHAQATGDELSRQLSLEEAGLFYTNIMTSEDIPLEVRDKV 240 241GMQFLAASLDMNQRGIYEGLRDAGFLDSMSFDDRRALNGLYEKSKAQTRAKESMATLRAD 300 301ADFQQPNANGAITDLAEVEAYSRGMVEEGRWSDAQAISFMTKAMTGLGNAQRMQGIMAAL 360 361EAGDINALHTLGTNVTEALEQWDKMQAANGSSLTDRLVQGTQLGLRLGTFPKTYGESVGS 420 421AVRMIQAAKEGEANPELVNTLNSIFEQVASAQEINPSAGNVMLSGIPEAEQGAVAWALKQ 480 481MKMGIAPAQALREFSANAEVVKQMDEFEKGQNTKAFKDNLGKQVNDKFVNNIFGRAWNML 540 541TGESDLSNNEAVLSMYRRATIDEANWLASDRKHAGLLTSDTGREALLEIAAANVRNRTIQ 600 601VGEGRNLKREGDLFSRRDSAPLILPRGTTAEQLFGTNDTETIGTVLAEQHKPVEGLLGYK 660 661SVVAFEYDRTSGSLLAVEYDENGVALDRTRVDPQAVGNEVLKRNADKLNAMRGAEYGANV 720 721KVSGTDIRMNGGNSAGMLKQDVFNWRKELAQFEAYRGEAYKDADGYSVGLGHYLGSGNAG 780 781AGTTVTPEQAAQWFAEDTDRALDQGVRLADELGVTNNASILGLAGMAFQMGEGRARQFRN 840 841TFQAIKDRNKEAFEAGVRNSKWYTQTPTGAEAFIKRMAPHFDTPSQIGVDWYSAATAE 898SEQ ID NO: 3 (Homo Sapiens cDNA, FLJ96367, highly similar to Homo sapiensbactericidal/permeability-increasing protein (BPI), mRNA: GenBank: AK315328.1)ORIGIN 1aggccttgag gttttggcag ctctggagga tgagagagaa catggccagg ggcccttgca 61acgcgccgag atgggcgtcc ctgatggtgc tggtcgccat aggcaccgcc gtgacagcgg 121ccgtcaaccc tggcgtcgtg gtcaggatct cccagaaggg cctggactac gccagccagc 181aggggacggc cgctctgcag aaggagctga agaggatcaa gattcctgac tactcagaca 241gctttaagat caagcatctt gggaaggggc attatagctt ctacagcatg gacatccgtg 301aattccagct tcccagttcc cagataagca tggtgcccaa tgtgggcctt aagttctcca 361tcagcaacgc caatatcaag atcagcggga aatggaaggc acaaaagaga ttcttaaaaa 421tgagcggcaa ttttgacctg agcatagaag gcatgtccat ttcggctgat ctgaagctgg 481gcagtaaccc cacgtcaggc aagcccacca tcacctgctc cagctgcagc agccacatca 541acagtgtcca cgtgcacatc tcaaagagca aagtggggtg gctgatccaa ctcttccaca 601aaaaaattga gtctgcgctt cgaaacaaga tgaacagcca ggtctgcgag aaagtgacca 661attctgtatc ctccgagctg caaccttatt tccagactct gccagtaatg accaaaatag 721attctgtggc tggaatcaac tatggtctgg tggcacctcc agcaaccacg gctgagaccc 781tggatgtaca gatgaagggg gagttttaca gtgagaacca ccacaatcca cctccctttg 841ctccaccagt gatggagttt cccgctgccc atgaccgcat cgtatacctg ggcctctcag 601actacttctt caacacagcc gggcttgtat accaagaggc tggggtcttg aagatgaccc 961ttagagatga catgattcca aaggagtcca aatttcgact gacaaccaag ttctttggaa 1021ccttcctacc tgaggtggcc aagaagtttc ccaacatgaa gatacagatc catgtctcag 1081cctccacccc gccacacctg tctgtgcagc ccaccggcct taccttctac cctgccgtgg 1141atgtccaggc ctttgccgtc ctccccaact cctccctggc ttccctcttc ctgattggca 1201tgcacacaac tggttccatg gaggtcagcg ccgaatccga caggcttgtt ggagagctca 1261agctggatag gctgctcctg gaactgaagc actcaaatat tggccccttc ccggttgaat 1321tgctgctgga tatcatgaac tacattgtac ccattcttgt gctgcccagg gttaacgaga 1381aactacagaa aggcttccct ctcccgacgc cggccagagt ccagctctac aacgtagtgc 1441ttcagcctca ccagaacttc ctgctgttcg gtgcagacgt tgtctataaa tgaSEQ ID NO: 4 (BPI; unnamed protein product [Homo sapiens]GenBank: BAG37729.1) ORIGIN 1mrenmargpc naprwaslmv lvaigtavta avnpgvvvri sqkgldyasq qgtaalgkel 61krikipdysd sfkikhlgkg hysfysmdir efqlpssqis mvpnvglkfs isnanikisg 121kwkaqkrflk msgnfdlsie gmsisadlkl gsnptsgkpt itcascsshi nsvhvhisks 181kvgwliqlfh kkiesalrnk mnsqvcekvt nscsselqpy fqtlpvmtki dsvaginygl 241vappattaet ldvqmkgefy senhhnpppf appvmefpaa hdrmvylgls dyffntaglv 301yqeagvkkmt lrddmipkes kfrlttkffg tflpevakkf pnmkiqihvs astpphlsvq 361ptgltfypav dvqafavlpn sslaslflig mhttgsmevs aesdrlvgel kldrillelk 421hsnigpfpve llldimnyiv pilvlprvne klqkgfplpt parvqlynvv lqphgnfllf 481gadvvykSEQ ID NO: 5 (P134 GP36 nucleic acid; first CHC13 test construct) 1ATGGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTGCTC 60 61AAGCGCAACGCGGACAAGCTGAATGCGATGCGGGGCGCCGAGTACCGTGCCAACGTCAAG 120 121GTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAGGAC 100 181GTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTATAAG 240 241GATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGGGCA 300 301GGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGCGCA 360 361CTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATCCTG 420 421GGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAACACC 480 481TTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGCAAG 540 541TGGTACACGCAGACGCCCACCGGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCACTTC 600 601GATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGTAA 654SEQ ID NO: 6 (24 Kda construct; translation product of SEQ ID NO: 5, 217 amino acids.N-terminal Met may be removed in a prokaryotic production host, so N-terminus ofprotein may begin with G.) 1MGVALDRTRVDPQAVGNEVLKRNADKLNAMRGAEYGANVKVSGTDIRMNGGNSAGMLKQD 60 61VFNWRKELAQFEAYRGEAYKDADGYSVGLGHYLGSGNAGAGTTVTPEQAAQWFAEDTDRA 120 121LDQGVRLADELGVTNNASILGLAGMAFQMGEGRARQFRNTFQAIKDRNKEAFEAGVRNSK 180 181WYTQTPTGAEAFIRKMAPHFDTPSQIGVDWYSAATAE 217SEQ ID NO: 7 [13 aa C terminal extesion attached for purification purposes onto24 Kda] 1 MGVALDRTRVDPQAVGNEVLKRNADKLNAMRGAEYGANVKVSGTDIRMNGGNSAGMLKQD60 61 VFNWRKELAQFEAYRGEAYKDADGYSVGLGHYLGSGNAGAGTTVTPEQAAQWFAEDTDRA 120121 LDQGVRLADELGVTNNASILGLAGMAFQMGEGRARQFRNTFQAIKDRNKEAFEAGVRNSK 180 181WYTQTPTGAEAFIKRMAPHFDTPSQIGVDWYSAATAEKLAAALEHHHHHH 230SEQ ID NO: 8 [encoding chimeric GP36 segment linked to BPI segment; P225]a. Construct nucleotide sequence: P225 1ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGT 60 61CATATGGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTG 120 121CTCAAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTC 180 181AAGGTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAG 240 241GACGTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTAT 300 301AAGGATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGG 360 361GCAGGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGC 420 421GCACTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATC 480 481CTGGGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAAC 540 541ACCTTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGC 600 601AAGTGGTACACGCAGACGCCCACCGGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCAC 660 661TTCGATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGCGCCGT 720 721CGCGCGTCCCTGATGGTGCTGGTCGCCATAGGCACCGCCGTGACAGCGGCCGTCAACCCT 780 781GGCGTCGTGGTCAGGCGCCGTCGCTAA 807Part 1 (1-66) is N terminal extension including poly His fox purification (22 aa)Part 2 (67-714) is GP36 muralytic fragment domain segment (aa 683 to 898; 217 aa)Part 3 (715-804) is linker (RRR) with BPI TMD domain segment (aa 16-39) With RRR Cterminus (33 aa)SEQ ID NO: 9 [vector seq-GP36 muralytic domain-RRR-BPI TMD-RRR; P225 polypeptide; Nterminus may have Met removed in a prokaryotic production host] 1MGHHHHHHHHHHSSGHIEGRHMGVALDRTRVDPQAVGNEVLKRNADKLNAMRGAEYGANV 60 61KVSGTDIRMNGGNSAGMLKQDVFNWRKELAQFEAYRGEAYKDADGYSVGLGHYLGSGNAG 120 121AGTTVTPEQAAQWFAEDTDRALDQGVRLADELGVTNNASILGLAGMAFQMGEGRARQFRN 180 181TFQAIKDRNKEAFEAGVRNSKWYTQTPTGAEAFIKRMAPHFDTPSQIGVDWYSAATAERR 240 241RASLMVLVAIGTAVTAAVNPGVVVRRRR 268SEQ ID NO: 10 P266 construct Nucleic acid:     1-6 =ATG (start codon) GGC: Bases generated due to cloning enzyme (NheI) site   7-24 = Sequence encoding 6Xhis tag  25-672 =Sequence encoding GP36CD sequence 673-681 =Sequence encoding linker arginines 682-753 = Sequence encoding BPI MTD754-762 = Sequence encoding terminal arginines 763-765 =TGA: Sequence encoding stop codon 1ATGGGCCATCATCATCATCATCATGGTGTAGCTCTTGATCGCACGCGGGTTGATCCCCAG 60 61GCAGTCGGCAACGAGGTGCTCAAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCC 120 121GAGTACGGTGCCAACGTCAAGGTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGT 180 181GCCGGCATGCTGAAGCAGGACGTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCT 240 241TACCGAGGGGAGGCGTATAAGGATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTG 300 301GGCAGTGGCAATGCTGGGGCAGGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTC 360 361GCCGAGGACACCGACCGCGCACTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTT 420 421ACGAACAATGCCTCTATCCTGGGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGT 480 481GCCCGGCAGTTCCGTAACACCTTCCAGGCGATCAAGGATCGCAACAAGGAAGCCATCGAG 540 541GCTGGTGTGCGAAACAGCRAGTGGTACACGCAGACGCCCAACCGGGCCGAGGCATTCATC 600 601AAGCGCATGGCGCCCCACTTCGATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCC 660 661GCAACAGCGGAGCGCCGTCGCGCGTCCCTGATGGTGCTGGTCGCCATAGGCACCGCCGTG 720 721ACAGCGGCCGTCAACCCTGGCGTCGTGGTCAGGCGCCGTCGCTGA 765SEQ ID NO: 11 P266 amino acid sequence (254 aa):       1 =M (start codon; removed by producing coli host)       2 =G: Amino acid generated due to cloning enzyme site     3-8 = 6Xhis tag  9-240 = GP36 Catalytic (muralytic) Domain sequence 241-243 =Linker arginines 228-251 = BPI TMD 252-254 = N-Terminal arginines[vector seq-his5-GP36 muralytic domain-RRR-BPI TMD-RRR; P266 polypeptide]1 MGHHHHHHGV ALDRTRVDPQ AVGNEVLKRN ADKLNAMRGA EYGANVKVSG TDIRMNGGNS 6061 AGMLKQDVFN WRKELAQFEA YRGEAYKDAD GYSVGLGHYL GSGNAGAGTT VTPEQAAQWF 120121 AEDTDRALDQ GVRLADELGV TNNASILGLA GMAFQMGEGR ARQFRNTFQA IKDRNKEAFE190 181AGVRNSKWYT QTPTGAEAFI KRMAPHFDTP SQIGVDWYSA ATAERRRASL MVLVAIGTAV 240241 TAAVNPGVVV RRRR 254SEQ ID NO: 12 Nuclectide sequence of P275 construct 1ATGGGCCATCATCATCATCATCATGGTGTAGCTCTTGATCGCACGCGGGTTGATCCCCAG 60 61GCAGTCGGCAACGAGGTGCTCAAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCC 120 121GAGTACGGTGCCAACGTCAAGGTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGT 180 181GCCGGCATGCTGAAGCAGGACGTGTTCAACTGGCGGAAGGAACTGGCTGAGTTCGAGGCT 240 241TACCGAGGGGAGGCGTATAAGGATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTG 300 301GGCAGTGGCAATGCTGGGGCAGGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTC 360 361GCCGAGGACACCGACCGCGCACTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTT 420 421ACGAACAATGCCTCTATCCTGGGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGT 480 481GCCCGGCAGTTCCGTAACACCTTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAG 540 541GCTGGTGTGCGAAACAGCAAGTGGTACACGCAGACGCCCAACCGGGCCGAGGCATTCATC 600 601AAGCGCATGGCGCCCCACTTCGATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCC 660 661GCAACAGCGGAGCGCCGTCGCGCGTCCCTGATGGAGCTGGACGCCAAAGGCACCGCCGTG 720 721ACAGCGGCCGTCAACCCTGGCGTCGTGGTCAGGCGCCGTCGCTGA 765SEQ ID NO: 13 P275 polypeptide construct; in BPI domain V232 to E; V234 to D; I236 toK 1 MGGHHHHHGV ALDRTRVDPQ AVGNEVLKRN ADKLNAMRGA EYGANVKVSG TDIRMNGGNS 6061 AGMLKDQVFN WRKELAQFEA YRGEAYKDAD GYSVGLGHYL GSGNAGAGTT VTPEQAAQWF 120121 AEDTDRALDQ GVRLADELGV TNNASILGLA GMAFQMGEGR ARQFRNTFQA IKDRNKEAFE180 181AGVRNSKWYT QTPTGAEAFI KRMAPHFDTP SQIGVDWYSA ATAERRRASL MELDAKGTAV 240241 TAAVNPGVVV RRRR 254SEQ ID NO: 14 GP36 MD - P134 Holin MTD chimera construct 1ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGT 60 61CATATGGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTG 120 121CTCAAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTC 100 181AAGGTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAG 240 241GACGTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTAT 300 301AAGGATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGG 360 361GCAGGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGC 420 421GCACTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATC 490 481CTGGGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAAC 540 541ACCTTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGC 600 601AAGTGGTACACGCAGACGCCCACCGGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCAC 660 661TTCGATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGCGCCGT 120 721CGCGAGATCGCCAGCCTCTGTGCTGCGGTACTCACCGCGCTCTACGTGGGCGCCCAGCTC 180 781ATCACCCTGCTCCGCCGTCGCTGA 804SEQ ID NO: 15 GP36 MD - P134 holin MTD polypeptide sequence 1MGHHHHHHHH HHSSGHIEGR HMGVALDRTR VDPQAVGNEV LKRNADKLNA MRGAEYGANV 60 61KVSGTDIRMN GGNSAGMLKQ DVFNWRKELA QFEAYRGEAY KDADGYSVGL GHYLGSGNAG 120121 AGTTVTPEQA AQWFAEDTDR ALDQGVRLAD ELGVTNNASI LGLAGMAFQM GEGRARQFRN180 181TFQAIKDRNK EAFEAGVRNS KWYTOTPTGA EAFIKRMAPH FDTPSQIGVD WYSAATAERR 240241 REIASLCAAV LTALYVGAQL ITLLRRR 267SEQ ID NO: 16 GP36 MD - Lipopolysaccharide binding proten MTD construct1 ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGT 60 61CATATGGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTG 120 121CTCAAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTC 180 181AAGGTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAG 240 241GACGTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTAT 300 301AAGGATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGG 360 361GCAGGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGC 120 421GCACTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATC 480 481CTGGGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGGCCGGCAGTTCCGTAAC 540 541ACCTTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGC 600 601AAGTGGTACACGCAGACGCCCACCGGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCAC 660 661TTCGATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGCGCCGT 720 721CGCTCCGACTCCTCCATCCGGGTCCAGGGCCGTTGGAGGTGCGCGCGTCATTCTTCAAA 780 761CTGCAGGGCTCCTTCGATGTCAGTGTCAAGGGCCGCCGTCGCTGA 825SEQ ID NO: 17 GP36 MD - LPS binding protein MTD construct polypeptide sequence1 MGHHHHHHHH HHSSGHIEGR HMGVALDRTR VDPQAVGNEV LKRNADKLNA MRGAEYGANV 6061 KVSGTDIRMN GGNSAGMLKQ DVFNWRKELA QFEAYRGEAY KDADGYSVGL GHYLGSGNAG 120121 AGTTVTPEQA AQWFAEDTDR ALDQGVRLAD ELGVTNNASI LGLAGMAFQM GEGRARQFRN180 181TFQAIKDRNK EAFEAGVRNS KWYTQTPTGA EAFIKRMAPH FDTPSQIGVD WYSAATAERR 240241 RSDSSTRVQG RWKVRASFFK LQGSFDVSVK GRRR 274SEQ ID NO: 18 GP36 MD - T4 phage, gp5 beta helix MTD chimera construct 1ATGGGTGTGGCCCTGGACCGCACGCGGCTTGATCCCCAGGCAGTCGGCAACGAGGTGCTC 60 61AAGCGCAACGC0GATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTCAAG 120 121GTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAGGAC 180 181GTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTATAAG 240 241GATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGGGCA 300 301CGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGCGCA 360 361CTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATCCTG 420 421GGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAACACC 480 481TTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGCAAG 540 541TGGTACACGCAGACGCCCAACCGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCACTTC 600 601GATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGAAGCTTTAT 660 661GTGCATACAATGGAAACTGAAAGCGGACATATTCAGGAATTTGATGATACCCCTGGGCAA 720 721GAACGATATAGATTAGTTCATCCAACTGGAACTTATGAAGAAGTATCACCATCAGGAAGA 780 781AGAACAAGAAAAACTGTTGATAATTTGTATGATATAACCAATGCTGATGGTAATTTTTTG 840 841GTAGCCGGTGATAAAAAGACTAACGTCGGTGGTTCAGAAATTTATTATAACATGGATAAT 900 901CGTTTACATCAAATCGATGGAAGCAATACAATATTTGTACGTGGAGACGAAACGAAAACT 960 961GTTGAAGGTAATGGAACTATCCTAGTTAAAGGTAATGTTACTATTATAGTTGAAGGTAAT 1020 1021GCTGACATTACAGTTAAAGGAGATGCTACCACTTTAGTTGAAGGAAATCAAACTAACACA 1080 1081GTAAATGGAAATCTTTCTTGGAAAGTTGCCGGGACAGTTGATTGGGATGTCGGTGGTGAT 1140 1141TGGACAGAAAAAATGGCATCTATGAGTTCTATTTCATCTGGTCAATACACAATTGATGGA 1200 1201TCGAGGATTGACATTGGCCTCGAGCACCACCACCACCACCACTAA 1245SEQ ID NO: 19 GP36 MD - T4 phage, gp5 beta helix MTD chimera polypeptide1 MGVALDRTRV DPQAVGNEVL KRNADKLNAM RGAEYGANVK VSGTDIRMNG GNSAGMLKQD 6061 VFNWREELAQ FEAYRGEAYK DADGYSVGLG HYLGSGNAGA GTTVTPEQAA QWFAEDTDRA 120121 LDQGVRLADE LGVTNNASIL GLAGMAFQMG EGRARQFRNT FQAIKDRNKE AFEAGVRNSK180 181WYTQTPNRAE AFIKRMAPHF DTPSQIGVDW YSAATAEKLY VHTMETESGH IQEFDDTPGQ 240241 ERYRLVHPTG TYEEVSPSGR RTRKTVDNLY DITNADGNFL VAGDKKTNVG GSEIYYNMDN300 301RLHQIDGSNT IFVRGDETKT VEGNGTILVK GNVTIIVEGN ADITVKGDAT TLVEGNQTNT 360361 VNGNLSWKVA GTVDWDVGGD WTEKMASMSS ISSGQYTIDG SRIDIGLEHH HHHH 414SEQ ID NO: 20 GP36 MD - S type pyocin outer membrane translocation (OMT) domainchimera construct 1ATGGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTGCTC 60 61AAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTCAAG 120 121GTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAGGAC 180 181GTGTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTATAAG 240 241GATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGGGCA 300 301GGTACTACAGTGACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGCGCA 360 361CTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATCCTG 420 421GGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAACACC 480 481TTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGCAAG 540 541TGGTACACGCAGACGCCCAACCGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCACTTC 500 601GATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGCAGAAGCTTCAA 660 661GCGTTGCAAGATGCTATTAAATTTACTGCCGACTTTTATAAGGAAGTAACTGAGAAATTT 720 721GGCGCACGAACATCGGAGATGGCGCGCCAACTGGCCGAAGGCGCCAGGGGGAAAAATATC 780 781AGGAGTTCGGCGGAAGCAATCAAGTCGTTTGAAAAGCACAAGGATGCGTTAAATAAAAAA 840 841CTTAGCCTTAAAGATAGGCAAGCCATTGCCAAAGCCTTTGATTCTCTAGACAAGCAGATG 900 901ATGGCGAAGAGCCTTGAGAAATTTAGCAAAGGCTTTGGAGTTGTAGGCAAAGCTATTGAC 960 961GCCGCCAGCCTGTACCAAGAGTTCAAGATATCTACGGAAACCGGGGACTGGAAACCATTC 1020 1021TTTGTAAAAATTGAAACACTAGCTGCTGGTGCGGCCGCCAGTTGGCTTGTGGGTATTGCA 1080 1081TTTGCCACGGCAACAGCCACTCCTATAGGCATTCTGGGGTTCGCACTGGTAATGGCAGTT 1140 1141ACCGGGGCCATGATTGACGAAGACCTTCTAGAAAAAGCAAACAATCTTGTAATATCCATT 1200 1201CTCGAGCACCACCACCACCACCACTAA 1227SEQ ID NO: 21 GP36 MD - S type pyocin (OMT) domain chimera polypeptide408 aa, Theoretical pI/Mw: 8.44 / 44543.37 1MGVALDRTRV DPQAVGNEVL KRNADKLNAM RGAEYGANVK VSGTDIRMNG GNSAGMLKQD 60 61VFNWRKELAQ FEAYRGEAYK DADGYSVGLG HYLGSGNAGA GTTVTPEQAA QWFAEDTDRA 120121 LDQGVRLADE LGVTNNASIL GLAGMAFQMG EGRARQFRNT FQAIKDRNKE AFEADVRNSK180 181WYTQTPNRAE AFIKRMAPHF DTPSQIGVDW YSAATAEKLQ ALQDAIKFTA DFYKEVTEKF 240241 GARTSEMARQ LAEGARGKNI RSSAEAIKSF EKHKDALNKK LSLKDRQAIA KAFDSLDKQM300 301MAKSLEKFSK GFGVVGKAID AASLYQEFKI STETGDWKPF FVKIETLAAG AAASWLVGIA 360361 FATATATPIG ILGFALVMAV TGAMIDEDLL EKANNLVISI LEHHHHHH 408SEQ ID NO: 22 GP36 MD - P22 Tail Spike protein MTD chimera construct 1ATGGGTGTGGCCCTGGACCGCACGCGGGTTGATCCCCAGGCAGTCGGCAACGAGGTGCTC 60 61AAGCGCAACGCGGATAAGCTGAATGCGATGCGGGGCGCCGAGTACGGTGCCAACGTCAAG 120 121GTCAGCGGCACGGACATTCGCATGAACGGGGGTAACAGTGCCGGCATGCTGAAGCAGGAC 180 181GTCTTCAACTGGCGGAAGGAACTGGCTCAGTTCGAGGCTTACCGAGGGGAGGCGTATAAG 240 241GATGCCGATGGTTATAGTGTGGGCCTGGGGCATTACCTGGGCAGTGGCAATGCTGGGGCA 300 301GGTACTACAGTCACGCCTGAGCAAGCCGCGCAGTGGTTCGCCGAGGACACCGACCGCGCA 360 361CTCGACCAGGGTGTGAGGTTGGCCGACGAGCTGGGCGTTACGAACAATGCCTCTATCCTG 420 421GGATTGGCCGGTATGGCCTTCCAGATGGGCGAAGGACGTGCCCGGCAGTTCCGTAACACC 450 481TTCCAGGCGATCAAGGATCGCAACAAGGAAGCCTTCGAGGCTGGTGTGCGAAACAGCAAG 540 541TGGTACACGCAGACGCCCAACCGGGCCGAGGCATTCATCAAGCGCATGGCGCCCCACTTC 600 601GATACACCGAGTCAAATCGGTGTCGATTGGTACAGCGCCGCAACAGCGGAGAAGCTTACA 660 661GACATCACTGCAAACGTAGTTGTTTCTAACCCTCGTCCAATCTTCACTGAATCCCGTTCG 720 721TTTAAAGCTGTTGCTAATGGGAAAATTTACATTGGTCAGATTGATACCGATCCGGTTAAT 780 781CCTGCCAATCAGATACCCGTATACATTGAAAATGAGGATGGCTCTCACGTCCAGATTACT 840 841CAGCCGCTAATTATCAACGCAGCCGGTAAAATCGTATACAACGGCCAACTGGTGAAAATT 900 901GTCACCGTTCAGGGTCATAGCATGGCTATCTATGATGCCAATGGTTCTCAGGTTGACTAT 960 961ATTGCTAACGTATTGAAGTACGATCCAGATCAATATTCAATAGAAGCTGATAAAAAATTT 1020 1021AAGTATTCAGTAAAATTATCAGATTATCCAACATTGCAGGATGCAGCATCTGCTGCGGTT 1080 1081GATGGCCTTCTTATCGATCGAGATTATAATTTTTATGGTGGAGAGACAGTTGATTTTGGC 1140 1141GGAAAGGTTCTGACTATAGAATGTAAAGCTAAATTTATAGGAGATGGAAATCTTATTTTT 1200 1201ACGAAATTAGGCAAAGGTTCCCGCATTGCCGGGGTTTTTATGGAAAGCACTACAACACCA 1260 1261TGGGTTATCAAGCCTTGGACGGATGACAATCAGTGGCTAACGGATGCCGCAGCGGTCGTT 1320 1321GCCACTTTAAAACAATCTAAAACTGATGGGTATCAGCCAACCGTAAGCGATTACGTTAAA 1380 1351TTCCCAGGAATAGAAACGTTACTCCCACCTAATGCAAAAGGGCAAAACATAACGTCTACG 1440 1441TTAGAAATTAGAGAATGTATAGGGGTCGAAGTTCATCGGGCTAGCGGTCTAATGGCTGGT 1500 1501TTTTTGTTTAGAGGGTGTCACTTCTGCAAGATGGTAGACGCCAATAATCCAAGCGGAGGT 1560 1561AAAGATGGCATTATAACCTTCGAAAACCTTAGCGGCGATTGGGGGAAGGGTAACTATGTC 1620 1621ATTGGCGGACGAACCAGCTATGGGTCAGTAAGTAGCGCCCAGTTTTTACGTAATAATGGT 1680 1681GGCTTTGAACGTGATGGTGGAGTTATTGGGTTTACTTCATATCGCGCTGGGGAGAGTGGC 1740 1741GTTAAAACTTGGCAAGGTACTGTGGGCTCGACAACCTCTCGCAACTATAATCTGCAATTC 1300 1801CGCGACTCGGTCGTTATTTACCCCGTATGGGACGGATTCGATTTAGGTGCTGACACTGAC 1860 1861ATGAATCCGGAGTTGGACAGGCCAGGGGACTACCCTATAACCCAATACCCACTGCATCAG 1920 1921TTACCCCTAAATCACCTGATTGATAATCTTCTGGTTCGCGGGGCGTTAGGTGTAGGTTTT 1980 1981GGTATGGATGGTAAGGGCATGTATGTGTCTAATATTACCGTAGAAGATTGCGCTGGGTCT 2340 2041GGCGCGTACCTACTCACCCACGAATCAGTATTTACCAATATAGCCATAATTGACACCAAT 2100 2101ACTAAGGATTTCCAGGCGAATCACATTTATATATCTGGGGCTTGCCGTGTGAACGGTTTA 2160 2161CGTTTAATTGGCATCCGCTCAACCGATGGGCAGGGTCTAACCATAGACGCCCCTAACTCT 2220 2221ACCGTAAGCGGTATAACCGGGATGGTAGACCCCTCTAGAATTAATGTTGCTAATTTGGCA 2280 2281GAAGAAGGGTTAGGTAATATCCGCGCTAATAGTTTCGGCTATGATAGCGCAGCGATTAAA 2340 2341CTGCGGATTCATAAGTTATCAAAGACATTAGATAGCGGAGCATTGTACTCCCACATTAAC 2400 2401GGGGGGGCCGGTTCTGGCTCAGCGTATACTCAACTTACTGCTATTTCAGGTAGCACACCT 2460 2461GACGCTGTATCATTAAAAGTTAACCACAAAGATTGCAGGGGGGCAGAGATACCATTTGTT 2520 2521CCTGACATCGCGTCAGATGATTTTATAAAGGATTCCTCATGTTTTTTGCCATATTGGGAA 2580 2581AATAATTCTACTTCTTTAAAGGCTTTAGTGAAAAAACCCAATGGAGAATTAGTTAGATTA 2640 2641ACCTTGGCAACACTTCTCGAGCACCACCACCACCACCACTAG 2682SEQ ID NO: 23 GP36 - P22 Tail Spike protein MTD polypeptide 1MGVALDRTRV DPQAVGNEVL KRNADKLNAM RGAEYGANVK VSGTDIRMNG GNSAGMLKQD 60 61VFNWRKELAQ FEAYRGEAYK DADGYSVGLG HYLGSGNAGA GTTVTPEQAA QWFAEDTDRA 120121 LDQGVRLADE LGVTNNASIL GLAGMAFQMG EGRARQFRNT FQAIKDRNKE AFEAGVRNSK180 181WYTQTPNRAE AFIKRMAPHF DTPSQIGVDW YSAATAEKLT DITANVVVSN PRPIFTESRS 243241 FKAVANGKIY IGQIDTDPVN PANQIPVYIE NEDGSHVQIT QPLIINAAGK IVYNGQLVKI300 301VTVQGHSMAI YDANGSQVDY IANVLKYDPD QYSIEADKKF KYSVKLSDYP TLQDAASAAV 360361 DGLLIDRDYN FYGGETVDFG GKVLTIECKA KFIGDGNLIF TKLGKGSRIA GVFMESTTTP420 421WVIKPWTDDN QWLTDAAAVV ATLKQSKTDG YQPTVSDYVK FRGIETLLPP NAKGQNITST 480481 LEIRECIGVE VRRASGLMAG FLFRGCHFCK MVDANNPSGG KDGIITFENL SGDWGKGNYV540 541IGGRTSYGSV SSAQFLRNNG GFERDGGVIG FTSYRAGESG VKTWQGTVGS TTSRNYNLQF 600601 RDSVVIYPVW DGFDLGADTD MNPELDRPGD YPITQYPLHQ LPLNHLIDNL LVRGALGVGF660 661GMDGKGMYVS NITVEDCAGS GAYLLTHESV FTNIAIIDTN TKDFQANQIY ISGACRVNGL 720721 RLIGIRSTDG QGLTIDAPNS TVSGITGMVD PSRINVANLA EEGLGNIRAN SFGYDSAATK780 781LRIHKLSKTL DSGALYSHIN GGAGSGSAYT QLTAISGSTP DAVSLKVNHK DCRGAEIPFV 840841 PDIASDDFIK DSSCFLPYWE NNSTSLKALV KKPNGELVRL TLATLLEHHH HHH 893

1. A chimeric polypeptide comprising: (i) a muralytic domain (MD) from avirion-associated muralytic enzyme or a variant thereof capable oflysing chloroform-treated Pseudomonas aeruginosa bacteria; and (ii) amembrane traversing domain (MTD) having a DAS profile between 1.5 and 3,and capable of traversing the outer membrane of Pseudomonas aeruginosabacteria.
 2. The chimeric polypeptide of claim 1, wherein 1-100 nmol ofthe MD lyses at least 50% of 10⁷ chloroform-treated Pseudomonasaeruginosa bacteria in a CFU drop assay.
 3. A chimeric polypeptide ofclaim 1, wherein: (i) the MD comprises a sequence of amino acids 737-875of SEQ ID NO:2, or a variant thereof capable of lysingchloroform-treated gram negative bacteria; and (ii) the MTD comprisesamino acids 16-39 of SEQ ID NO:4, or a variant thereof with a DASprofile between 1.2 and 2.6.
 4. The chimeric polypeptide of claim 1,wherein 1-100 nmol of the MTD transverses the membrane of at least 50%of 10⁷ Pseudomonas aeruginosa bacteria.
 5. The chimeric polypeptide ofclaim 1, wherein said MD comprises a sequence of amino acids 737-875 ofSEQ ID NO:2, or a variant with 1, 2, or 3 of the methionine amino acidssubstituted with non-methionine amino acids.
 6. The chimeric polypeptideof claim 1, wherein said MD comprises a sequence of amino acids 683-889of SEQ ID NO:2, or a variant with 1-7 of the methionine amino acidssubstituted with non-methionine amino acids.
 7. The chimeric polypeptideof claim 1, wherein the MTD comprises a sequence of amino acids 16-39 ofSEQ ID NO:4, or a variant with 1-6 hydrophobic amino acids substitutedwith amino acids with a hydropathy score of −2 or lower.
 8. The chimericpolypeptide of claim 7, wherein V20, V22, and I24 of SEQ ID NO:4 aresubstituted with amino acids with a hydropathy score of −2 or lower. 9.The chimeric polypeptide of claim 1, wherein the DAS profile of the MTDis under 2.5.
 10. The chimeric polypeptide of claim 1, wherein the DASprofile of the chimeric polypeptide is under 2.5.
 11. The chimericpolypeptide of claim 1, wherein the MTD and MD are joined by a linkercomprising 3-5 positively charged amino acids.
 12. The chimericpolypeptide of claim 1, wherein the MD comprises a sequence at least 90%identical to amino acids 737-875 of SEQ ID NO:2 and the MTD comprises asequence at least 80% identical to amino acids 16-39 of SEQ ID NO:4. 13.The chimeric polypeptide of claim 1, wherein the chimeric polypeptidecomprises a sequence having at least 90% identity to a sequence selectedfrom: SEQ ID NO:9, SEQ ID NO: 11, and SEQ ID NO:
 13. 14. The chimericpolypeptide of claim 1, wherein the chimeric polypeptide comprises thesequence of SEQ ID NO:
 11. 15. The chimeric polypeptide of claim 1,wherein the chimeric polypeptide comprises a sequence of SEQ ID NO: 13.16. The chimeric polypeptide of claim 1, wherein the chimericpolypeptide is attached to polyethylene glycol.
 17. A pharmaceuticalcomposition comprising the chimeric polypeptide of claim 1 and apharmaceutically acceptable excipient.
 18. A method of inhibitingbacterial cell growth in an individual comprising administering thepharmaceutical composition of claim 17 to the individual.
 19. The methodof claim 18, wherein the bacteria are selected from the group consistingof Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli,Klebsiella pneumoniae, Acinetobacter baumanii, Salmonella typhimurium,Salmonella infantis, Shigella, Proteus mirabilis, and Burkholderiathailandensis.
 20. An antibacterial formulation comprising the chimericpolypeptide of claim 1 and an agent that reduces oxidation.
 21. A methodof inhibiting bacterial cell growth in an environment comprisingapplying the chimeric polypeptide of claim 1 to the environment.
 22. Themethod of claim 21, wherein the bacteria are selected from the groupconsisting of Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichiacoli, Klebsiella pneumoniae, Acinetobacter baumanii, Salmonellatyphimurium, Salmonella infantis, Shigella, Proteus mirabilis, andBurkholderia thailandensis. 23-32. (canceled)